Red-shifted opsin molecules and uses thereof

ABSTRACT

The invention, in some aspects relates to compositions and methods for altering cell activity and function and the use of light-activated ion pumps (LAIPs).

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/295,750, filed Nov. 14, 2011, now pending, which claims the benefitunder 35 U.S.C. §119(e) of U.S. Provisional application Ser. No.61/413,431 filed Nov. 13, 2010, the disclosure of each of which isincorporated by reference herein in its entirety.

GOVERNMENT INTEREST

This invention was made with government support under Grant Nos. R01DA029639, R01 NS067199 and RC1 MH088182 awarded by the NationalInstitutes of Health. The government has certain rights in theinvention.

FIELD OF THE INVENTION

The invention, in some aspects relates to compositions and methods foraltering cell activity and function and the use of light-activated ionpumps.

BACKGROUND OF THE INVENTION

Altering and controlling cell membrane and subcellular region ionpermeability has permitted examination of characteristics of cells,tissues, and organisms. Light-driven pumps and channels have been usedto silence or enhance cell activity and their use has been proposed fordrug screening, therapeutic applications, and for exploring cellular andsubcellular function.

Molecular-genetic methods for preparing cells that can be activated(e.g., depolarized) or inactivated (e.g., hyperpolarized) by specificwavelengths of light have been developed (see, for example, Han, X. andE. S. Boyden, 2007, PLoS ONE 2, e299). It has been identified that thelight-activated cation channel channelrhodopsin-2 (ChR2), and thelight-activated chloride pump halorhodopsin (Halo/NpHR), whentransgenically expressed in cell such as neurons, make them sensitive tobeing activated by blue light, and silenced by yellow light,respectively (Han, X. and E. S. Boyden, 2007, PLoS ONE 2(3): e299;Boyden, E. S., et. al., 2005, Nat Neurosci. 2005 September; 8(9):1263-8.Epub 2005 Aug. 14.). Previously identified light-activated pumps andchannels have been restricted to activation by particular wavelengths oflight, thus limiting their usefulness.

SUMMARY OF THE INVENTION

The invention, in part, relates to isolated light-activated ion pump(LAIP) polypeptides and methods of their preparation and use. Theinvention also includes isolated nucleic acid sequences that encodelight-driven ion pumps of the invention as well as vectors andconstructs that comprise such nucleic acid sequences. In addition, theinvention in some aspects includes expression of light-activated ionpump polypeptides in cells, tissues, and organisms as well as methodsfor using the light-activated ion pumps to alter cell and tissuefunction and for use in diagnosis and treatment of disorders.

The invention, in part, also relates to methods for adjusting thevoltage potential of cells, subcellular regions, or extracellularregions. Some aspects of the invention include methods of incorporatingat least one nucleic acid sequence encoding a light-driven ion pump intoat least one target cell, subcellular region, or extracellular region,the ion pump functioning to change transmembrane potential in responseto a specific wavelength of light. Exposing an excitable cell thatincludes an expressed light-driven ion pump of the invention to awavelength of light that activates the pump, results in hyperpolarizingvoltage change in the excitable cell. By contacting a cell that includesa LAIP of the invention with particular wavelengths of light in the redspectrum, the cell is hyperpolarized strongly enough to shift thevoltage potential of the excitable cell to a level that silences spikeactivity of the excitable cell. A plurality of light-activated ion pumpsactivated by different wavelengths of light may be used to achievemulti-color excitable cell silencing.

Methods and LAIPs of the invention can be used for neural silencing,that is, hyperpolarizing a neuron to prevent it from depolarizing,spiking, or otherwise signaling (e.g., to release neurotransmitters). ALAIP of the invention can be used in conjunction with one or more otherlight-activated membrane proteins for multi-color neural silencing(e.g., Ace or Mac, and Halo), expressing them in different populationsof cells in a tissue or in a dish, and the illuminating them each withdifferent colors of light.

The ability to optically perturb, modify, or control cellular functionoffers many advantages over physical manipulation mechanisms, such asspeed, non-invasiveness, and the ability to easily span vast spatialscales from the nanoscale to macroscale. One such approach is anopto-genetic approach, in which heterologously expressed light-activatedmembrane polypeptides such as a LAIP of the invention, are used to moveions with various spectra of light.

According to some aspects of the invention, isolated light-activated ionpump polypeptides that when expressed in an excitable cell and contactedwith a red light silences the excitable cell, wherein the polypeptidesequence of the light-activated ion pump comprises a wild-type ormodified halomicrobium or haloarcula halorhodopsin polypeptide sequence,are provided. In certain embodiments, the modified haloarcula orhalomicrobium halorhodopsin polypeptide sequence comprises the aminoacid sequence of a wild-type haloarcula or halomicrobium halorhodopsin,respectively, with one, two, or more amino acid modifications. Incertain embodiments, the modified haloarcula halorhodopsin polypeptidehas the amino acid sequence of Halo57 (SEQ ID NO:2); Gene4 (SEQ IDNO:4); Gene58 (SEQ ID NO:6); Gene56 (SEQ ID NO:8); Gene55 (SEQ IDNO:10); and the modified halomicrobium halorhodopsin polypeptide has theamino acid sequence of Gene54 (SEQ ID NO:12), with one, two, or moreamino acid modifications. In some embodiments, the modified haloarculaor halomicrobium halorhodopsin polypeptide sequence comprises one ormore of: a) a K→R, K→H, or K→Q substitution at an amino acid residuecorresponding to amino acid 200 of the amino acid sequence of Halo57(SEQ ID NO:2); b) a T→S substitution at an amino acid residuecorresponding to amino acid 111 of the amino acid sequence of Halo57(SEQ ID NO:2); c) a T→S substitution at an amino acid residuecorresponding to amino acid 203 of the amino acid sequence of Halo57(SEQ ID NO:2); or d) a K→Q+W→F double substitution at the amino acidresidues corresponding to amino acid 200 and 214, respectively, of theamino acid sequence of Halo57 (SEQ ID NO:2). In some embodiments, themodified halobacterium halorhodopsin polypeptide sequence comprises aK→R modification and a W→F modification at the amino acid residuescorresponding to amino acid 200 and 214, respectively, of the amino acidsequence of Halo57 (SEQ ID NO:2). In certain embodiments, the modifiedhaloarcula halorhodopsin polypeptide sequence is the sequence set forthas SEQ ID NO:26. In some embodiments, the light-activated ion pumpexpressed is activated by contacting the pump with light having awavelength in the range from about 450 nm to about 690 nm. In someembodiments, the wavelength of the red light is in the range of about620 nm to about 690 nm.

According to another embodiment, cells that include any embodiment of anaforementioned aspect of the invention are provided. In someembodiments, the light-activated ion pump is activated and the cellhyperpolarized when the light-activated ion pump is contacted with lightunder suitable conditions for hyperpolarization. In certain embodiments,the light-activated ion pump is activated and the cell silenced when thelight-activated ion pump is contacted with red light under suitableconditions for silencing. In some embodiments, the cell is an excitablecell. In some embodiments, the cell is a non-excitable cell. In certainembodiments, the cell is a mammalian cell. In some embodiments, the cellis in vitro, ex vivo, or in vivo. In some embodiments, the cell alsoincludes one, two, three, four, or more additional light-activated ionpumps, wherein at least one, two, three, four, or more of the additionallight-activated ion pumps is activated by contact with light having anon-red light wavelength.

According to another aspect of the invention an isolated nucleic acidsequence that encodes any embodiment of an aforementioned isolatedlight-activated ion pump polypeptide, is provided. In some embodiments,the nucleic acid sequence is a mammalian codon-optimized DNA sequence.In certain embodiments, the light-activated ion pump encoded by thenucleic acid sequence is expressed in the cell.

According to another aspect of the invention, vectors that include anyembodiment of an aforementioned nucleic acid sequence are provided. Insome embodiments, the nucleic acid sequence is operatively linked to apromoter sequence. In some embodiments, the vector also includes one,two, or more nucleic acid signal sequences operatively linked to thenucleic acid sequence encoding the light-activated ion pump. In certainembodiments, the vector is a plasmid vector, cosmid vector, viralvector, or an artificial chromosome.

According to another aspect of the invention, cells that include anyembodiment of an aforementioned vector, are provided. In someembodiments, the cell also includes one, two, three, four, or moreadditional light-activated ion pumps, wherein at least one, two, three,four, or more of the additional light-activated ion pumps is activatedby contact with light having a non-red light wavelength.

According to another aspect of the invention, methods of hyperpolarizinga cell are provided. The methods include expressing in a cell anembodiment of an aforementioned isolated light-activated ion pumppolypeptide, and contacting the isolated light-activated ion pump with alight that activates the isolated light-activated ion pump andhyperpolarizes the cell. In some embodiments, the light is a red lightthat hyperpolarizes and silences the cell. In certain embodiments, thecell is in vivo, ex vivo, or in vitro. In some embodiments, the methodalso includes delivering to the cell a nucleic acid sequence thatencodes the isolated light-activated ion pump. In some embodiments, thenucleic acid sequence is delivered by means of a vector. In someembodiments, the modified haloarcula or halomicrobium halorhodopsinpolypeptide comprises the amino acid sequence of a wild-type haloarculaor halomicrobium halorhodopsin polypeptide, respectively with one, two,or more amino acid modifications. In certain embodiments, the modifiedhaloarcula or halomicrobium halorhodopsin polypeptide sequence comprisesone or more of: a) a K→R, K→H, or K→Q substitution at an amino acidresidue corresponding to amino acid 200 of the amino acid sequence ofHalo57 (SEQ ID NO:2); b) a T→S substitution at an amino acid residuecorresponding to amino acid 111 of the amino acid sequence of Halo57(SEQ ID NO:2); c) a T→S substitution at an amino acid residuecorresponding to amino acid 203 of the amino acid sequence of Halo57(SEQ ID NO:2); or d) a K→Q+W→F double substitution at the amino acidresidues corresponding to amino acid 200 and 214, respectively, of theamino acid sequence of Halo57 (SEQ ID NO:2). In some embodiments, themodified haloarcula or halomicrobium halorhodopsin polypeptide sequencecomprises a K→R modification and a W→F modification at amino acidresidues corresponding to amino acids 200 and 214, respectively, of theamino acid sequence of Halo57 (SEQ ID NO:2). In some embodiments, themodified haloarcula halorhodopsin polypeptide sequence is the sequenceset forth as SEQ ID NO:26. In certain embodiments, the light-activatedion pump is activated and the cell silenced when the light-activated ionpump is contacted with a red light under suitable conditions forsilencing. In some embodiments, the light-activated ion pump isactivated by contact with light having a wavelength from about 450 nm toabout 690 nm. In certain embodiments, the red light has a wavelengthfrom about 620 nm to about 690 nm. In some embodiments, the cell is anervous system cell, a neuron, a cardiac cell, a circulatory systemcell, a visual system cell, an auditory system cell, a hemoglobin-richcell, or a muscle cell. In some embodiments, wherein the cell is anexcitable cell. In some embodiments, the cell is a non-excitable cell.In certain embodiments, the cell is a mammalian cell. In someembodiments, the cell also includes one, two, three, or more additionallight-activated ion pumps, wherein at least one, two, three, four, ormore of the additional light-activated ion pumps is activated by contactwith light having a non-red light wavelength. In some embodiments, thecell is in a subject and hyperpolarizing the cell diagnoses or assistsin a diagnosis of a disorder in the subject. In some embodiments, thecell is in a subject and silencing the cell diagnosis or assists in adiagnosis of a disorder in the subject. In certain embodiments, the cellis in a subject and hyperpolarizing the cell treats a disorder in thesubject. In some embodiments, the cell is in a subject and silencing thecell treats a disorder in the subject.

According to yet another aspect of the invention, methods of identifyingan effect of a candidate compound on a cell are provided. The methodsinclude contacting a test cell comprising an isolated light-activatedion pump of any embodiment of an aforementioned aspect, with a lightunder conditions suitable to activate the ion pump and hyperpolarize thetest cell; contacting the test cell with a candidate compound; andidentifying the presence or absence of a change in the hyperpolarizationor in a hyperpolarization-mediated cell characteristic in the test cellcontacted with the light and the candidate compound compared to thehyperpolarization or the hyperpolarization-mediated cell characteristic,respectively, in a control cell contacted with the light and notcontacted with the candidate compound; wherein a change in thehyperpolarization or the hyperpolarization-mediated cell characteristicin the test cell compared to the control identifies an effect of thecandidate compound on the test cell. In some embodiments, the effect ofthe candidate compound is an effect on the hyperpolarization of the testcell. In some embodiments, the effect of the candidate compound is aneffect on a hyperpolarization-mediated cell characteristic in the testcell. In certain embodiments, the hyperpolarization mediated cellcharacteristic is a hyperpolarization-activated conductance. In someembodiments, the hyperpolarization-activated conductance is the resultof a T-type calcium channel activity, a BK channel activity, or an I_hcurrent. In some embodiments, the hyperpolarization mediated-cellcharacteristic is cell silencing. In some embodiments, the modifiedhaloarcula or halomicrobium halorhodopsin polypeptide sequence comprisesthe amino acid sequence of a wild-type haloarcula or halomicrobiumhalorhodopsin polypeptide, respectively, with one, two, or more aminoacid modifications. In certain embodiments, the modified haloarcula orhalomicrobium halorhodopsin polypeptide sequence comprises one or moreof: a) a K→R, K→H, or K→Q substitution at an amino acid residuecorresponding to amino acid 200 of the amino acid sequence of Halo57(SEQ ID NO:2); b) a T→S substitution at an amino acid residuecorresponding to amino acid 111 of the amino acid sequence of Halo57(SEQ ID NO:2); c) a T→S substitution at an amino acid residuecorresponding to amino acid 203 of the amino acid sequence of Halo57(SEQ ID NO:2); or d) a K→Q+W→F double substitution at the amino acidresidues corresponding to amino acid 200 and 214, respectively, of theamino acid sequence of Halo57 (SEQ ID NO:2). In some embodiments, themodified haloarcula or halomicrobium halorhodopsin polypeptide sequencecomprises a K→R modification and a W→F modification at the amino acidresidues corresponding to amino acid 200 and 214, respectively, of theamino acid sequence of Halo57 (SEQ ID NO:2). In some embodiments, themodified haloarcula halorhodopsin polypeptide sequence is the sequenceset forth as SEQ ID NO:26. In certain embodiments, the method alsoincludes characterizing the change identified in the hyperpolarizationor the hyperpolarization-mediated cell characteristic. In someembodiments, the method also includes contacting the light-activated ionpump with a red light, wherein the red light silences the cell. In someembodiments, the light-activated ion pump is activated by contacting thepump with light having a wavelength from about 450 nm to about 690 nm.In some embodiments, the red-light has a wavelength from about 620 nm toabout 690 nm. In certain embodiments, the test cell is a nervous systemcell, a neuron, a cardiac cell, a circulatory system cell, a visualsystem cell, an auditory system cell, a hemoglobin-rich cell, or amuscle cell. In some embodiments, the test cell is an excitable cell. Insome embodiments, the test cell is a non-excitable cell. In someembodiments, the test cell is a mammalian cell. In certain embodiments,the cell also includes one, two, three, or more additionallight-activated ion pumps, wherein at least one, two, three, four, ormore of the additional light-activated ion pumps is activated by contactwith light having a non-red light wavelength.

According to another aspect of the invention, methods of treating adisorder in a subject are provided. The methods include administering toa subject in need of such treatment, a therapeutically effective amountof a light-activated ion pump any embodiment of an aforementionedaspect, to treat the disorder. In some embodiments, the light-activatedion pump is administered in the form of a cell, wherein the cellexpresses the light-activated ion pump; or in the form of a vector,wherein the vector comprises a nucleic acid sequence encoding thelight-activated ion pump and the administration of the vector results inexpression of the light-activated ion pump in a cell in the subject. Insome embodiments, the method also includes contacting the cell withlight and activating the light-activated ion pump in the cell. Incertain embodiments, the method also includes contacting the cell withred light and activating and silencing the light-activated ion pump inthe cell. In some embodiments, the cell is a nervous system cell, aneuron, a cardiac cell, a circulatory system cell, a visual system cell,an auditory system cell, a hemoglobin-rich cell, or a muscle cell. Insome embodiments, the vector further comprises a signal sequence. Insome embodiments, the vector further comprises a cell-specific promoter.In certain embodiments, the disorder is a neurological disorder, avisual system disorder, a circulatory system disorder, a musculoskeletalsystem disorder, or an auditory system disorder.

According to another aspect of the invention, isolated light-activatedion pump polypeptides that when expressed in an excitable cell andcontacted with a red light silences the excitable cell are provided andthe polypeptide sequence of the light-activated ion pump comprises amodified Halobacteriaceae halorhodopsin polypeptide sequence, and themodified halorhodopsin polypeptide sequence comprises one or more of a)a K→R, K→H, or K→Q substitution at an amino acid residue correspondingto amino acid 200 of the amino acid sequence of Halo57 (SEQ ID NO:2); b)a T→S substitution at an amino acid residue corresponding to amino acid111 of the amino acid sequence of Halo57 (SEQ ID NO:2); c) a T→Ssubstitution at an amino acid residue corresponding to amino acid 203 ofthe amino acid sequence of Halo57 (SEQ ID NO:2); or d) a K→Q+W→F doublesubstitution at the amino acid residues corresponding to amino acid 200and 214, respectively, of the amino acid sequence of Halo57 (SEQ IDNO:2). In some embodiments, the modified halorhodopsin polypeptidesequence comprises a K→R substitution at an amino acid residuecorresponding to amino acid 200 of the amino acid sequence of Halo57(SEQ ID NO:2) and further comprises a W→F substitution at the amino acidresidue corresponding to amino acid 214 of the amino acid sequence ofHalo57 (SEQ ID NO:2). In some embodiments, the sequence of thelight-activated ion pump polypeptide is the sequence set forth as SEQ IDNO:26.

According to another aspect of the invention, cells that include anyembodiment of an aforementioned isolated light-activated ion pumppolypeptide are provided. In some embodiments, the cell is an excitablecell. In certain embodiments, the cell is a non-excitable cell. In someembodiments, the cell is a mammalian cell. In some embodiments, the cellis in vitro, ex vivo, or in vivo. In certain embodiments, the cell alsoincludes one, two, three, four, or more additional light-activated ionpumps, wherein at least one, two, three, four, or more of the additionallight-activated ion pumps is activated by contact with light having anon-red light wavelength.

According to another aspect of the invention, nucleic acid sequencesthat encode any embodiment of an aforementioned isolated light-activatedion pump polypeptide are provided. According to yet another aspect ofthe invention, vectors that include any embodiment of an aforementionednucleic acid sequence are provided. In some embodiments, the vector alsoincludes one, two, or more nucleic acid signal sequences operativelylinked to the nucleic acid sequence encoding the light-activated ionpump.

According to another aspect of the invention, cells that include anyembodiment of an aforementioned vector are provided. In someembodiments, the cell is an excitable cell, and optionally a mammaliancell. In some embodiments, the cell is a non-excitable cell. In certainembodiments, the cell is in vitro, ex vivo, or in vivo. In someembodiments, the cell also includes one, two, three, four, or moreadditional light-activated ion pumps, wherein at least one, two, three,four, or more of the additional light-activated ion pumps is activatedby contact with light having a non-red light wavelength.

According to yet another aspect of the invention, methods ofhyperpolarizing a cell are provided. The methods include contacting acell comprising any embodiment of an aforementioned isolatedlight-activated ion pump polypeptide with a light under conditionssuitable to activate the ion pump and hyperpolarize the cell. In someembodiments, the light is a red light that hyperpolarizes and silencesthe cell. In certain embodiments, the method also includes delivering tothe cell a nucleic acid sequence that encodes the isolatedlight-activated ion pump. In some embodiments, the cell is a mammaliancell. In some embodiments, the cell is in a subject and hyperpolarizingthe cell diagnoses or assists in a diagnosis of a disorder in thesubject. In certain embodiments, the cell is in a subject and silencingthe cell diagnosis or assists in a diagnosis of a disorder in thesubject. In some embodiments, the cell is in a subject andhyperpolarizing the cell treats a disorder in the subject. In someembodiments, the cell is in a subject and silencing the cell treats adisorder in the subject. In some embodiments, the sequence of thelight-activated ion pump polypeptide the sequence set forth as SEQ IDNO:26.

According to another aspect of the invention, methods of identifying aneffect of a candidate compound on a cell are provided. The methodsinclude a) contacting a test cell comprising any embodiment of anaforementioned isolated light-activated ion pump polypeptide with alight under suitable conditions to activate the ion pump andhyperpolarize the test cell; b) contacting the test cell with acandidate compound; and c) identifying the presence or absence of achange in the hyperpolarization or in a hyperpolarization-mediated cellcharacteristic in the test cell contacted with the light and thecandidate compound compared to the hyperpolarization or thehyperpolarization-mediated cell characteristic, respectively, in acontrol cell contacted with the light and not contacted with thecandidate compound; wherein a change in the hyperpolarization or thehyperpolarization-mediated cell characteristic in the test cell comparedto the control indicates an effect of the candidate compound on the testcell. In certain embodiments, the method also includes characterizing achange identified in the hyperpolarization or thehyperpolarization-mediated cell characteristic. In some embodiments, themethod also includes contacting the light-activated ion pump with a redlight and silencing the cell. In some embodiments, the sequence of thelight-activated ion pump polypeptide the sequence set forth as SEQ IDNO:26.

According to yet another aspect of the invention, methods of treating adisorder in a subject are provided. The methods include, administeringto a subject in need of such treatment, a therapeutically effectiveamount of any embodiment of an aforementioned light-activated ion pumppolypeptide to treat the disorder. In some embodiments, thelight-activated ion pump is administered in the form of a cell, whereinthe cell expresses the light-activated ion pump or in the form of avector, wherein the vector comprises a nucleic acid sequence encodingthe light-activated ion pump and the administration of the vectorresults in expression of the light-activated ion pump in a cell in thesubject. In certain embodiments, the method also includes contacting thecell with light and activating the light-activated ion pump in the cell.In some embodiments, the method also includes contacting the cell withred light and activating and silencing the light-activated ion pump inthe cell. In some embodiments, the cell is a nervous system cell, aneuron, a cardiac cell, a circulatory system cell, a visual system cell,an auditory system cell, a hemoglobin-rich cell, an integumentary systemcell, or a muscle cell. In some embodiments, the vector furthercomprises a signal sequence. In certain embodiments, the vector furthercomprises a cell-specific promoter. In some embodiments, the disorder isa neurological disorder, a visual system disorder, a circulatory systemdisorder, or an auditory system disorder. In some embodiments, thesequence of the light-activated ion pump polypeptide the sequence setforth as SEQ ID NO:26.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing Photocurrent normalized action spectra forHalo57, ArchT and Mac, demonstrating that Halo57 (the Halobacteriumsalinarum (strain shark)/Halobacterium halobium (strain shark) gene forhalorhodopsin) is significantly red-light shifted relative to otheropsins.

FIG. 2 is a graph of light power versus photocurrent for various LAIPsand other molecules. The figure demonstrates the higher photocurrentachieved by Halo57 mutant and Halo57 mutant-Kir2.1 than others tested.

FIG. 3 is an illustration showing the amino acid sequence alignment ofrhodopsin sequences for: Halo57 (SEQ ID NO:2); Gene4 (SEQ ID NO:4);Gene58 (SEQ ID NO:6); Gene56 (SEQ ID NO:8); Gene55 (SEQ ID NO:10);Gene54 (SEQ ID NO:12) and Halo (SEQ ID NO:14).

FIG. 4A and FIG. 4B present two graphs of results relating to hemoglobinabsorption. FIG. 4A shows the trough of hemoglobin absorption and showsresults indicating that oxygen-bound hemoglobin absorbs 24 times morelight at 593 nm illumination than at 660 nm, near its trough ofabsorbance. FIG. 4B shows results indicating that Halo57 redshift, whichallows illumination at the hemoglobin absorption trough.

FIG. 5A and FIG. 5B present two histograms showing (photo)current forHalo, Halo57 and single and double mutants of Halo57. Screen data showsoutward photocurrents (FIG. 5A) and wildtype-normalized photocurrents(FIG. 5B) for Halo and Halo57 mutants, measured by whole-cellpatch-clamp of HEK293FT cells under screening illumination conditions(575±25 nm, 3.7 mW/mm2). Data are mean and s.e. The FIG. 5A histogramillustrates that Halo57 (the Halobacterium salinarum (strainshark/Halobacterium halobium (strain shark) gene for halorhodopsin)produced orders of magnitude more photocurrent than Halo whenilluminated at 660 nm (7 mW/mm²). FIG. 5B is a histogram showing thatthe photocurrents for the Halobacterium salinarum (strainshark/Halobacterium halobium (strain shark) halorhodopsin can be furtherimproved by the K200R+W214F double mutation.

FIG. 6A and FIG. 6B include two histograms showing Halo versus Halo57photocurrent comparisons. FIG. 6A shows photocurrent results for Halo(wild-type) and Halo that includes the single mutations: (K215R) and(W229F) and the double mutation [(K215R)+(W229F] and shows photocurrentresults for Halo57 (wild-type) and Halo57 that includes the singlemutations (K200R) and (W214F) and the double mutation [(K200R)+(W214F)].The position of the Halo sequence mutations correspond to the positionsof the mutations in the Halo57 sequence. FIG. 6B showswildtype-normalized photocurrents for Halo versus Halo57. In FIG. 6B,the first bar of each pair is Halo, and the second bar is Halo57. All ofthe values were normalized relative to the wildtype (mutant/wildtype).The experiment demonstrated the significantly different effect of thesubstitutions on Halo and Halo57 photocurrents. The left-most pair ofbars shows Halo (left) and Halo57 (right) wild-type normalized tothemselves. The second pair of bars from left show that K215Rsubstitution dropped Halo photocurrents but the correspondingsubstitution in Halo57 (K200R) boosted Halo57 photocurrent byapproximately 70%. The third set of bars from left show that W229F(equivalent=W214F in Halo57) lowered both Halo and Halo57, and finallythe far right pair of bars shows that the double mutation significantlylowered Halo photocurrent and substantially boosted Halo57 photocurrent.

FIG. 7 is a histogram showing the photocurrent effects obtained usingsignal sequences to boost membrane trafficking of light-activated ionpumps of the invention, as assessed in primary hippocampal mouse neuronculture via whole-cell patch claim. FIG. 7 provides screen data showingoutward photocurrents for Halo57 mutant with different appended celltrafficking signal sequences, measured by whole-cell patch-clamp ofcultured neurons under screening illumination conditions (575±25 nm,14.2 mW/mm²). Data are mean and s.e. Each bar demonstrates the differentamounts of photocurrent generated when illuminated with 570/50 nm light.

FIG. 8 is an image of traces showing the average spike rate of a cellthat includes a heterologous light-activated ion pump of the invention.The figure illustrates results of an in vivo implementation contactingthe light-activated ion pump with a light at 655 nm. The top trace is anextracellular recording showing the cell silencing during the fiveseconds of light contact, and the bottom trace shows the averagereduction in spike frequency over a total of seven trials.

FIG. 9 shows a histogram illustrating effects of mutagenesis of residuesin Halo57. The mutation is a double substitution K200R+W214F. Theresults shown are from combined trafficking and mutation studies. Acombination of protein trafficking enhancements with directedmutagenesis resulted in extremely powerful red-light drivable neuralsilencers. The expressed “ss” sequence includes the amino acid sequenceset forth as SEQ ID NO:16. The “ER2” sequence included the amino acidsequence set forth herein as SEQ ID NO: 20. The “ss-prl” sequenceincluded the amino acid sequences of SEQ ID NO:16 and the prolactinsignal sequence set forth herein as SEQ ID NO:18. The “ss-ER2” sequenceincluded the amino acid sequence of SEQ ID NO:16 and SEQ ID NO:18. The“prl-ER2” sequence included the amino acid sequence of SEQ ID NO: 18 andSEQ ID NO:20. The “ss-prl-ER2” sequence included the amino acid sequenceof SEQ ID NO:16 and SEQ ID NO:18); and SEQ ID NO:20. The “Kir2.1”sequence included the amino acid sequence of SEQ ID NO:22 and SEQ IDNO:20.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 is the mammalian codon-optimized DNA sequence that encodesHalo57, the gene for the Halobacterium salinarum (strain shark)halorhodopsin:

atgaccgccgtgagcaccacagccactaccgtgctgcaggccacacagagcgacgtgctgcaggagatccagtccaacttcctgctgaatagctccatctgggtgaacattgctctggccggagtggtcatcctgctgtttgtggccatggggagggatctggaatcccctagagctaagctgatctgggtggccacaatgctggtgccactggtgtctatttctagttacgctggactggccagtgggctgactgtgggcttcctgcagatgccacctggacacgctctggccggacaggaggtgctgagcccatggggccggtatctgacatggactttctccactcccatgatcctgctggctctgggactgctggccgacaccgatattgccagcctgttcaccgccatcacaatggacattggcatgtgcgtgacaggactggccgctgccctgatcactagctcccatctgctgcgctgggtgttctacggaatttcttgtgctttctttgtggccgtgctgtatgtgctgctggtgcagtggccagctgatgctgaggctgctgggaccagtgaaatctttggcactctgaagattctgaccgtggtgctgtggctggggtaccctatcctgtgggctctgggctctgagggagtggccctgctgagtgtgggagtgaccagctggggatactccggactggacatcctggctaaatacgtgttcgcctttctgctgctgagatgggtggctgccaatgaaggcacagtgtctgggagtggaatgggaatcgggtccggaggagctgctccagccgacgat.

SEQ ID NO: 2 is the amino acid sequence of Halo57, the gene for theHalobacterium salinarum (strain shark) halorhodopsin:

MTAVSTTATTVLQATQSDVLQEIQSNFLLNSSIWVNIALAGVVILLFVAMGRDLESPRAKLIWVATMLVPLVSISSYAGLASGLTVGFLQMPPGHALAGQEVLSPWGRYLTWTFSTPMILLALGLLADTDIASLFTAITMDIGMCVTGLAAALITSSHLLRWVFYGISCAFFVAVLYVLLVQWPADAEAAGTSEIFGTLKILTVVLWLGYPILWALGSEGVALLSVGVTSWGYSGLDILAKYVFAFLLLRWVAANEGTVSGSGMGIGSGGAAPADD.

SEQ ID NO:3 is the mammalian codon-optimized DNA sequence that encodesGene4, the gene for Haloarcula marismortui cruxhalorhodopsinhalorhodopsin:

Atgacagccgccagcaccaccgccaccaccgtgctgcaggccacacagtccgacgtgctgcaggagatccagagcaacttcctgctgaactccagcatctgggtgaacattgccctggccggcgtggtgatcctgctgtttgtggccatgggccgcgacctggaaagcccccgcgccaagctgatttgggtggccacaatgctggtgcccctggtgtccatcagcagctatgccggactggccagcggactgaccgtgggatttctgcagatgccccccggccacgccctggccggccaggaggtgctgtccccctggggccggtacctgacatggacattctccacccctatgatcctgctggccctgggactgctggccgatacagacatcgcctctctgttcaccgccatcaccatggacatcgggatgtgcgtgaccggactggccgccgccctgatcaccagctcccacctgctgcgctgggtgttctacggcatctcttgcgcctttttcgtggccgtgctgtacgtgctgctggtgcagtggcccgccgacgccgaggccgccggcaccagcgagatcttcggcacactgaagattctgacagtggtgctgtggctgggatacccaatcctgtgggccctgggctctgagggcgtggccctgctgagcgtgggagtgacctcttggggctacagcggactggacattctggccaagtacgtgttcgccttcctgctgctgaggtgggtggccgccaatgaaggaacagtgtctgggtccggcatgggcatcggctccgggggcgccacacctgccgacgac.

SEQ ID NO:4 is the amino acid sequence of Gene4, the gene for Haloarculamarismortui cruxhalorhodopsin halorhodopsin:

MTAASTTATTVLQATQSDVLQEIQSNFLLNSSIWVNIALAGVVILLFVAMGRDLESPRAKLIWVATMLVPLVSISSYAGLASGLTVGFLQMPPGHALAGQEVLSPWGRYLTWTFSTPMILLALGLLADTDIASLFTAITMDIGMCVTGLAAALITSSHLLRWVFYGISCAFFVAVLYVLLVQWPADAEAAGTSEIFGTLKILTVVLWLGYPILWALGSEGVALLSVGVTSWGYSGLDILAKYVFAFLLLRWVAANEGTVSGSGMGIGSGGATPADD.

SEQ ID NO:5 is the mammalian codon-optimized DNA sequence that encodesHalo58), the gene for Halobacterium salinarum (strain port)halorhodopsin:

atgaccgccgcttccaccacagctactaccatgctgcaggccacacagtctgacgtgctgcaggagatccagagtaacttcctgctgaatagctccatctgggtgaacattgctctggccggggtggtcatcctgctgtttgtggccatgggcagggatatcgaatctcctagagctaagctgatttgggtggccacaatgctggtgccactggtgagcatctctagttacgctgggctggcctccggactgactgtgggattcctgcagatgccacctggacacgctctggccggacaggaggtgctgtctccatggggccggtatctgacatggactttcagtactcccatgatcctgctggctctgggactgctggccgacaccgatattgccagcctgttcaccgccatcacaatggacattggaatgtgcgtgacagggctggccgctgccctgatcactagctcccatctgctgcgctgggtgttctacggaatttcttgtgctttctttgtggccgtgctgtatgtgctgctggtgcagtggccagctgatgctgaggctgctggcaccagcgaaatctttggaactctgaagattctgaccgtggtgctgtggctggggtaccctatcctgtgggctctgggaagcgagggagtggccctgctgtccgtgggagtgacatcttggggctacagtggactggacattctggctaaatacgtgttcgcctttctgctgctgagatgggtggctgccaatgaaggagccgtgtctgggagtggaatgagcatcgggtccggaggagctgctccagccgacgat.

SEQ ID NO:6 is the amino acid sequence of Halo58 (also referred toherein as Gene58), the gene for Halobacterium salinarum (strain port)halorhodopsin:

MTAASTTATTMLQATQSDVLQEIQSNFLLNSSIWVNIALAGVVILLFVAMGRDIESPRAKLIWVATMLVPLVSISSYAGLASGLTVGFLQMPPGHALAGQEVLSPWGRYLTWTFSTPMILLALGLLADTDIASLFTAITMDIGMCVTGLAAALITSSHLLRWVFYGISCAFFVAVLYVLLVQWPADAEAAGTSEIFGTLKILTVVLWLGYPILWALGSEGVALLSVGVTSWGYSGLDILAKYVFAFLLLRWVAANEGAVSGSGMSIGSGGAAPADD.

SEQ ID NO:7 is the mammalian codon optimized DNA sequence of Gene56,which encodes the Haloarcula sinaiiensis (ATCC 33800) halorhodopsin:

atgctgcaggagatccagtctaacttcctgctgaatagctccatctgggtgaacattgctctggccggagtggtcatcctgctgtttgtggccatggggagggacctggaaagtcctagagctaagctgatctgggtggccaccatgctggtgccactggtgagcatttctagttacgctggactggcctccggactgacagtgggcttcctgcagatgccacctggacacgctctggccggacaggaggtgctgtctccatggggccggtatctgacctggacattcagtacacccatgatcctgctggctctgggactgctggccgacactgatattgcttctctgtttactgccatcaccatggacattggcatgtgcgtgactggactggccgctgccctgatcaccagctcccatctgctgcgctgggtgttctacggaattagctgtgctttctttgtggccgtgctgtatgtgctgctggtgcagtggccagctgatgctgaggctgctgggacttccgaaatctttggcaccctgaagattctgacagtggtgctgtggctggggtaccctatcctgtgggctctgggctctgagggagtggccctgctgagtgtgggcgtgacaagctgggggtactccggcctggatatcctggctaaatacgtgttcgcctttctgctgctgagatgggtggccacaaatgaaggcaccgtgagcgggagtggaatgggaatcgggtccggaggagctgctccagccgacgat.

SEQ ID NO:8 is the amino acid sequence of Gene56, which is a Haloarculasinaiiensis (ATCC 33800) halorhodopsin:

MLQEIQSNFLLNSSIWVNIALAGVVILLFVAMGRDLESPRAKLIWVATMLVPLVSISSYAGLASGLTVGFLQMPPGHALAGQEVLSPWGRYLTWTFSTPMILLALGLLADTDIASLFTAITMDIGMCVTGLAAALITSSHLLRWVFYGISCAFFVAVLYVLLVQWPADAEAAGTSEIFGTLKILTVVLWLGYPILWALGSEGVALLSVGVTSWGYSGLDILAKYVFAFLLLRWVATNEGTVSGSGMGIGS GGAAPADD.

SEQ ID NO:9 is the DNA sequence of Gene55, which encodes Haloarculacaliforniae (ATCC 33799) halorhodopsin:

Atgaacatcgctctggccggagtggtcatcctgctgttcgtggctatgggaagggacctggagtcccctagagctaagctgatctgggtggccaccatgctggtgccactggtgtctattagctcctacgctggactggccagtgggctgacagtgggctttctgcagatgccacctggacacgctctggccggacaggaagtgctgagcccatggggccggtatctgacctggacattctccacacccatgatcctgctggctctgggactgctggccgacactgatattgcttctctgtttactgccatcaccatggacattggcatgtgcgtgactggactggccgctgccctgatcacctctagtcatctgctgcgctgggtgttctacggaatttcttgtgctttctttgtggccgtgctgtatgtgctgctggtgcagtggccagctgatgctgaggctgctgggactagtgaaatctttggcaccctgaagattctgacagtggtgctgtggctggggtaccctatcctgtgggctctgggcagcgagggagtggccctgctgtccgtgggagtgacatcttgggggtacagtggcctggatattctggctaaatacgtgttcgcctttctgctgctgagatgggtggccacaaatgaaggcactgtgagcgggtccggaatgggaatcgggagcggaggagctgccccagccgacgat.

SEQ ID NO:10 is the amino acid sequence of Gene55, which is theHaloarcula californiae (ATCC 33799) halorhodopsin:

MNIALAGVVILLFVAMGRDLESPRAKLIWVATMLVPLVSISSYAGLASGLTVGFLQMPPGHALAGQEVLSPWGRYLTWTFSTPMILLALGLLADTDIASLFTAITMDIGMCVTGLAAALITSSHLLRWVFYGISCAFFVAVLYVLLVQWPADAEAAGTSEIFGTLKILTVVLWLGYPILWALGSEGVALLSVGVTSWGYSGLDILAKYVFAFLLLRWVATNEGTVSGSGMGIGSGGAAPADD.

SEQ ID NO:11 is the DNA sequence of Gene54, which encodes Halomicrobiummukohataei DSM 12286 halorhodopsin:

Atgtccgccaccacaactctgctgcaggctactcagtctgaggctgtgaccgccatcgaaaacgacgtgctgctgagctcctctctgtgggctaatgtggctctggccggcctggctatcctgctgttcgtgtatatgggaaggaacgtggaggctccaagagccaagctgatttggggagccaccctgatgatccccctggtgagtattagtagctatctgggactgctgagcggactgacagtgggcttcatcgaaatgcctgctggacacgctctggccggagaggaagtgatgagtcagtggggcaggtacctgacttgggccctgtccaccccaatgatcctgctggctctgggactgctggccgacgtggatattggggacctgttcgtggtcatcgccgctgatattggaatgtgcgtgacagggctggccgctgccctgatcacttcctcttacggcctgcggtgggccttttatctggtgtcttgtgctttctttctggtggtgctgtacgctatcctggtggagtggccacagagcgccaccgctgctgggacagacgaaattttcggcacactgcgcgccctgactgtggtgctgtggctgggatatcctatcatttgggctgtgggaatcgagggactggctctggtgcagtccgtgggcctgaccagttggggatacagcgccctggatattggggccaaatatctgttcgcttttctgctgctgcggtgggtggctgccaatcaggacgtggtggggcagccctccctggatacccattctgaaggcacagctcctgccgacgat.

SEQ ID NO:12 is the amino acid sequence of Gene54, which isHalomicrobium mukohataei DSM 12286 halorhodopsin:

MSATTTLLQATQSEAVTAIENDVLLSSSLWANVALAGLAILLFVYMGRNVEAPRAKLIWGATLMIPLVSISSYLGLLSGLTVGFIEMPAGHALAGEEVMSQWGRYLTWALSTPMILLALGLLADVDIGDLFVVIAADIGMCVTGLAAALITSSYGLRWAFYLVSCAFFLVVLYAILVEWPQSATAAGTDEIFGTLRALTVVLWLGYPIIWAVGIEGLALVQSVGLTSWGYSALDIGAKYLFAFLLLRWVAANQDVVGQPSLDTHSEGTAPADD.

SEQ ID NO:13 is the DNA sequence of Halo, which encodes Natromonaspharaonis halorhodopsin:

Atgactgagaccctcccacccgtgactgaaagcgccgtcgctctgcaagcagaggttacccagcgggagctgttcgagttcgtcctcaacgaccccctcctggcttctagcctctacatcaacattgctctggcaggcctgtctatactgctgttcgtcttcatgaccaggggactcgatgaccctagggctaaactgattgcagtgagcacaattctggttcccgtggtctctatcgcttcctacactgggctggcatctggtctcacaatcagtgtcctggaaatgccagctggccactttgccgaagggagttctgtcatgctgggaggcgaagaggtcgatggggttgtcacaatgtggggtcgctacctcacctgggctctcagtacccccatgatcctgctggcactcggactcctggccggaagtaacgccaccaaactcttcactgctattacattcgatatcgccatgtgcgtgaccgggctcgcagctgccctcaccaccagcagccatctgatgagatggttttggtatgccatctcttgtgcctgctttctggtggtgctgtatatcctgctggtggagtgggctcaggatgccaaggctgcagggacagccgacatgtttaatacactgaagctgctcactgtggtgatgtggctgggttaccctatcgtttgggcactcggcgtggagggaatcgcagttctgcctgttggtgtgacaagctggggctactccttcctggacattgtggccaagtatatttttgcctttctgctgctgaattatctgacttccaatgagtccgtggtgtccggctccatactggacgtgccatccgccagcggcacacctgccgatgac.

SEQ ID NO:14 is the amino acid sequence of Halo, which is Natromonaspharaonis halorhodopsin:

MTETLPPVTESAVALQAEVTQRELFEFVLNDPLLASSLYINIALAGLSILLFVFMTRGLDDPRAKLIAVSTILVPVVSIASYTGLASGLTISVLEMPAGHFAEGSSVMLGGEEVDGVVTMWGRYLTWALSTPMILLALGLLAGSNATKLFTAITFDIAMCVTGLAAALTTSSHLMRWFWYAISCACFLVVLYILLVEWAQDAKAAGTADMFNTLKLLTVVMWLGYPIVWALGVEGIAVLPVGVTSWGYSFLDIVAKYIFAFLLLNYLTSNESVVSGSILDVPSASGTPADD.

SEQ ID NO:15 is the DNA sequence of the ‘ss’ signal sequence fromtruncated MHC class I antigen:

gtcccgtgcacgctgctcctgctgttggcagccgccctggctccgactca gacgcgggcc.

SEQ ID NO:16 is the amino acid sequence of the ‘ss’ signal sequence fromtruncated MHC class I antigen:

MVPCTLLLLLAAALAPTQTRA.

SEQ ID NO:17 is the DNA sequence of a prolactin signal sequence (alsoreferred to herein as “Prl”:

gacagcaaaggttcgtcgcagaaagggtcccgcctgctcctgctgctggtggtgtcaaatctactcttgtgccagggtgtggtctccacccccgtc.

SEQ ID NO:18 is the amino acid sequence of a prolactin signal sequence(also referred to herein as “Prl”:

MDSKGSSQKGSRLLLLLVVSNLLLCQVVS.

SEQ ID NO:19 is the DNA sequence of the ER export sequence (alsoreferred to herein as ER2”:

ttctgctacgagaatgaagtg.

SEQ ID NO:20 is the amino acid sequence of the ER export sequence (alsoreferred to herein as “ER2”:

FCYENEV.

SEQ ID NO:21 is the DNA sequence of KGC, which is a C terminal exportsequence from the potassium channel Kir2.1:

Aaatccagaattacttctgaaggggagtatatccctctggatcaaataga catcaatgtt.

SEQ ID NO:22 is the amino acid sequence of KGC, which is a C terminalexport sequence from the potassium channel Kir2.1:

KSRITSEGEYIPLDQIDINV.

SEQ ID NO:23 is the DNA sequence of a Halo-GFP fusion gene:

atgactgagaccctcccacccgtgactgaaagcgccgtcgctctgcaagcagaggttacccagcgggagctgttcgagttcgtcctcaacgaccccctcctggcttctagcctctacatcaacattgctctggcaggcctgtctatactgctgttcgtcttcatgaccaggggactcgatgaccctagggctaaactgattgcagtgagcacaattctggttcccgtggtctctatcgcttcctacactgggctggcatctggtctcacaatcagtgtcctggaaatgccagctggccactttgccgaagggagttctgtcatgctgggaggcgaagaggtcgatggggttgtcacaatgtggggtcgctacctcacctgggctctcagtacccccatgatcctgctggcactcggactcctggccggaagtaacgccaccaaactcttcactgctattacattcgatatcgccatgtgcgtgaccgggctcgcagctgccctcaccaccagcagccatctgatgagatggttttggtatgccatctcttgtgcctgctttctggtggtgctgtatatcctgctggtggagtgggctcaggatgccaaggctgcagggacagccgacatgtttaatacactgaagctgctcactgtggtgatgtggctgggttaccctatcgtttgggcactcggcgtggagggaatcgcagttctgcctgttggtgtgacaagctggggctactccttcctggacattgtggccaagtatatttttgcctttctgctgctgaattatctgacttccaatgagtccgtggtgtccggctccatactggacgtgccatccgccagcggcacacctgccgatgaccgaccggtagtagcagtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtaca agtaa.

SEQ ID NO:24 is the amino acid sequence of a Halo-GFP fusionpolypeptide:

MTETLPPVTESAVALQAEVTQRELFEFVLNDPLLASSLYINIALAGLSILLFVFMTRGLDDPRAKLIAVSTILVPVVSIASYTGLASGLTISVLEMPAGHFAEGSSVMLGGEEVDGVVTMWGRYLTWALSTPMILLALGLLAGSNATKLFTAITFDIAMCVTGLAAALTTSSHLMRWFWYAISCACFLVVLYILLVEWAQDAKAAGTADMFNTLKLLTVVMWLGYPIVWALGVEGIAVLPVGVTSWGYSFLDIVAKYIFAFLLLNYLTSNESVVSGSILDVPSASGTPADDRPVVAVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK.

SEQ ID NO:25 is the amino acid sequence of a Halo-GFP fusion polypeptide

MTETLPPVTESAVALQAEVTQRELFEFVLNDPLLASSLYINIALAGLSILLFVFMTRGLDDPRAKLIAVSTILVPVVSIASYTGLASGLTISVLEMPAGHFAEGSSVMLGGEEVDGVVTMWGRYLTWALSTPMILLALGLLAGSNATKLFTAITFDIAMCVTGLAAALTTSSHLMRWFYAISCACFLVVLYILLVEWAQDAKAAGTADMFNTLKLLTVVMWLGYPIVWALGVEGIAVLPVGVTSWGYSFLDIVAKYIFAFLLLNYLTSNESVVSGSILDVPSASGTPADDRPVVAVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK.

SEQ ID NO:26 is the amino acid sequence of Halo57 with K200R and W214Fmutations.

MTAVSTTATTVLQATQSDVLQEIQSNFLLNSSIWVNIALAGVVILLFVAMGRDLESPRAKLIWVATMLVPLVSISSYAGLASGLTVGFLQMPPGHALAGQEVLSPWGRYLTWTFSTPMILLALGLLADTDIASLFTAITMDIGMCVTGLAAALITSSHLLRWVFYGISCAFFVAVLYVLLVQWPADAEAAGTSEIFGTLRILTVVLWLGYPILFALGSEGVALLSVGVTSWGYSGLDILAKYVFAFLLLRWVAANEGTVSGSGMGIGSGGAAPADD.

SEQ ID NO:27 is an amino acid sequence of a prolactin signal sequence:

DSKGSSQKGSRLLLLLVVSNLLLCQGVVSTPV.

DETAILED DESCRIPTION

The invention in some aspects relates to the expression in cells oflight-driven ion pump polypeptides that can be activated by contact withone or more pulses of light, which results in strong hyperpolarizationand silencing of the cell. Light-activated pumps of the invention, alsoreferred to herein as light-activated ion pumps (LAIPs) can be expressedin specific cells, tissues, and/or organisms and used to control cellsin vivo, ex vivo, and in vitro in response to pulses of light of asuitable wavelength.

When expressed in a cell (e.g., an excitable or non-excitable cell), aLAIP of the invention can be activated by contacting the cell with alight having a wavelength between about 450 nm and 690 nm (e.g., using asingle photon process—multiphoton may be higher). Although activationcan occur across the range of wavelengths from 450 nm through 690 nm,the strength of the hyperpolarization in the excitable cell (e.g., themagnitude of the voltage deflection) differs depending on the wavelengthof light in the range. Contact with light having a wavelength from about450 nm to below about 600 nm can hyperpolarize an excitable cell thatincludes an LAIP of the invention but the strength of the depolarizationmay be insufficient to silence the cell. The strength of thehyperpolarization can be tuned by contacting a LAIP of the inventionwith a desired wavelength of light. For example, for a weakerhyperpolarization, a LAIP-containing cell or tissue may be contactedwith a light having a wavelength at the lower end of the 450 nm to 690nm wavelength range. To have a stronger hyperpolarization effect, thecell may be contacted with a light having a longer wavelength light.Thus, the strength of hyperpolarization can be tuned using variouswavelengths of light.

Contact with a light in the red spectrum more strongly hyperpolarizesthe excitable cell and the hyperpolarization by light in the redspectrum range of wavelengths is sufficient to silence the excitablecell. For example, contact with light in a wavelength range such asbetween 620 nm and 690 nm, 640 nm and 690 nm, 660 nm and 670 nm, 660 nmand 690 nm, 670 nm and 690 nm, or 680 nm and 690 nm hyperpolarizes thecell at a level sufficient to silence the cell. Exemplary wavelengths oflight that may be used to silence a cell expressing a LAIP of theinvention, include wavelengths from at least about 620 nm, 625 nm, 630nm, 635 nm, 640 nm, 645 nm, 650 nm, 655 nm, 660 nm, 665 nm, 670 nm, 675nm, 680 nm, 685 nm, to about 690 nm, including all wavelengthstherebetweeen. Red-light cell silencing may be obtained in excitablecells in which one or more LAIPs of the invention are expressed.

As used herein, the terms “silence” or “silenced” used in the context ofcells means that an excitable cell in which the ability to initiateaction potential (also referred to as a spike) is substantially reducedor eliminated in the cell. For example, for initiation of an actionpotential, a cell at a baseline voltage of about −65 mV receives aninput signal that shifts cell's voltage up by up to approximately 15,20, 30, or 40 millivolts (mV). When the change in voltage is sufficientto reach the cell's spike initiation voltage threshold an actionpotential (e.g. a spike) results. When a cell is hyperpolarized byactivating a LAIP of the invention with light, the cell voltage becomesmore negative than the baseline level, and an incoming signal may or maynot be sufficient to raise the cell's voltage sufficiently to reach thethreshold and trigger an action potential in the cell. Thus, atwavelengths of light at the low end of the 450 nm to 690 nm wavelengthrange, a cell can be hyperpolarized but may not be silenced. It has beendiscovered that by contacting a cell expressing a LAIP of the inventionwith a red light, the cell's voltage may drop to a level that preventsan input signal from raising the cell voltage to the to the thresholdnecessary to trigger an action potential, and the cell is silenced.

It will be understood that resting voltages and voltages changesinitiated by light contact may differ depending on whether the cellexpressing an LAIP of the invention is in vitro, in vivo, and may alsoon the type of cell and its normal resting voltage. For example, incells in vivo, there may be feedback mechanisms that can limit thevoltage deflection, even though the cell is still silenced. Thus,changes in voltages may be assessed based on differences, not asabsolute values, in part because not all excitable cells have the sameresting membrane potential. For example, cardiac cells are far morehyperpolarized than cortical neurons, which are more hyperpolarized thanPurkinje neurons, which are more hyperpolarized than HEK and CHO cells.One skilled in the art will recognize how to assess voltages changes andcell silencing using standard methods.

In some embodiments, the presence of LAIPs in one, two, three, or more(e.g. a plurality) of cells in a tissue or organism, can result insilencing of the single cell or the plurality of cells by contacting theLAIPs with red light of suitable wavelength. Upon activation with asuitable wavelength for cell silencing, LAIPs of the invention may be upto 90%, 95%, or 100% effective in silencing a cell or plurality of cellsin which LAIPs are expressed.

In exemplary implementations, the invention comprises methods forpreparing and using genes encoding LAIPs of the invention that have nowbeen identified. The invention, in part, also includes isolated nucleicacids comprising sequences that encode LAIPs of the invention as well asvectors and constructs that comprise such nucleic acid sequences. Insome embodiments the invention includes expression of polypeptidesencoded by the nucleic acid sequences, in cells, tissues, and organisms.Also included in some aspects of the invention are methods ofcombinatorial optimization of genes encoding LAIPs through novel geneclasses, targeted protein site-directed mutagenesis, and potent proteintrafficking sequences. The resultant gene products, when expressed ingenetically targeted cells, allow the powerful hyperpolarization ofcellular voltage in response to pulses of light. These pumps can begenetically-expressed in specific cells (e.g., using a virus or othervector) and then used to control cells in intact organisms (includinghumans) as well as in in vitro and ex vivo cells in response to pulsesof light.

The magnitude of the current that can be pumped into cells expressingthe pumps encoded by sequences of the invention upon exposure to lowlight powers of long-wavelength visible light is significantly improvedfrom existing pumps (e.g., Halo/NpHR). The ion pumps of the inventionhave a red-shifted activation spectra that is unique from the activationspectra of other light-activated pumps and channels (e.g. Halo/NpHR,arch, Mac, ChR2, etc.) (see for example, FIG. 1).

It has been identified that LAIPs of the invention are activated andsilence cells at different wavelengths than some previously identifiedlight-activated ion pumps. Thus LAIPs of the invention can be used ineither alone, using a selective light spectrum for activation and/orcell silencing and can also be used in combination with otherlight-activated ion pumps that utilize different wavelength of light foractivation and/or cell silencing, thus allowing two, three, four, ormore different wavelengths of light to be used to hyperpolarize and/orsilence different sets of cells in a tissue or organism by expressingpumps with different activation spectra and/or different silencingspectra in different cells and then illuminating the tissue and/ororganism with the appropriate wavelengths of light to hyperpolarizeand/or silence the cells.

Use of ion pumps as described herein, permits multiple colors of lightto be used to alter the physiology of different sets of cells in thesame tissue, by expressing polypeptide ion pumps with differentactivation spectra genetically in different cells, and then illuminatingthe cells and/or tissue with different colors of light. Additionally,the low light power requirement allows the silencing of large tissuevolumes, and allows long distance optical silencing, which may be usedto advantage when targeting organs or tissues that are difficult tosurgically access. Additionally, low light power requirement preventstissue damage from light-caused heating. FIG. 2 shows light power levelsof various LAIPs of the invention.

Specific ranges of wavelengths of light useful to activate ion pumps ofthe invention are provided and described herein. It will be understoodthat a light of appropriate wavelength for activation and will have apower and intensity appropriate for activation. It is well known in theart that light pulse duration, intensity, and power are parameters thatcan be altered when activating a pump with light. Thus, one skilled inthe art will be able to adjust power, intensity appropriately when usinga wavelength taught herein to activate a LAIP of the invention. Abenefit of a red-light activated LAIP of the invention, may be theability to “tune” its response using an appropriate illuminationvariables (e.g., wavelength, intensity, duration, etc.) to activate thechannel. Methods of adjusting illumination variables and assessingvoltage changes in cells are well-known in the art and representativemethods can be found in publications such as: Chow et al. Nature 2010,Jan. 7; 463(7277):98-102; Han et al. Front Mol Neurosci. 2009; 2:12.Epub 2009 Aug. 27; Tang et al. J Neurosci. 2009 Jul. 8; 29(27):8621-9,each of which is incorporated herein by reference. Thus, it is possibleto utilize a narrow range of one or more illumination characteristics toactivate a LAIP of the invention. This may be useful to illuminate aLAIP that is co-expressed with one or more other light activated pumpsthat can be illuminated with a different set of illumination parametersfor their activation. Thus, permitting controlled activation of a mixedpopulation of light-activated pumps.

Taxonomy and Sequence Sources

In particular, the present invention includes, in part, the expressionand use of a novel class of LAIPs to hyperpolarize excitable cells andcan also be expressed and used to alter ion transport into non-excitablecells. In some non-limiting embodiments of the invention one or morenewly identified LAIPs may be expressed in cells. Some LAIPs of theinvention have amino acid sequences derived from halorhodopsins that arenaturally expressed in the genus Haloarcula or halomicrobium, or othermembers of the Halobacteriaceae family. Haloarcula, Halomicrobium, andother members of the Halobacteriaceae family are extreme halophilicarchaeons and are found in saline environment such as saline lakes,salterns, and some soils. Halorhodopsins of the haloarcula genus arealso known as cruxhalorhodopsins. Some embodiments of the inventioninclude isolated nucleic acid and/or amino acid halorhodopsin sequences,for example, from haloarcula or halomicrobium or other halobacteriaceaesequences, which may be wild-type or modified sequences, and methods fortheir use.

Microbial halorhodopsins of the haloarcula genus, include, but are notlimited to the Halobacterium salinarum (strain shark), which is alsoreferred to as Halobacterium halobium (strain shark) gene forhalorhodopsin, the Halobacterium salinarum (strain port), which is alsoreferred to as Halobacterium halobium (strain port) gene forhalorhodopsin, and the Haloarcula marismortui ATCC 43049 gene forhalorhodopsin. One skilled in the art will be able to identifyadditional Halobacteriaceae halorhodopsin sequences with sufficientamino acid sequence homology to halorhodopsin sequences such as Halo57,Gene4, Gene58, set forth herein, to be able to apply methods of theinvention using the additional halorhodopsin sequences.

LAIPs of the invention are transmembrane polypeptides that use lightenergy to move ions and protons into the cell in which they areexpressed, thus altering the cell's membrane potential. A non-limitingexample of an ion that can be moved into a cell the using a LAIP of theinvention is a chloride ion, potassium ion, and/or a sodium ion. LAIPsof the invention can be activated by light, either sustained light orlight pulses and can inhibit or eliminate initiation of actionpotentials in the cells in which they are expressed.

The wild-type and modified Halobacteriaceae halorhodopsin nucleic acidand amino acid sequences used in aspects and methods of the inventionare “isolated” sequences. As used herein, the term “isolated” used inreference to a polynucleotide, nucleic acid sequence or polypeptidesequence of a halorhodopsin, it means a polynucleotide, nucleic acidsequence, or polypeptide sequence that is separate from its nativeenvironment and present in sufficient quantity to permit itsidentification or use. Thus, an isolated polynucleotide, nucleic acidsequence, or polypeptide sequence of the invention is a polynucleotide,nucleic acid sequence, or polypeptide sequence that is not part of, orincluded in its native host. For example, a nucleic acid or polypeptidesequence may be naturally expressed in a cell or organism of a member ofthe haloarcula genus, but when the sequence is not part of or includedin a haloarcula cell or organism it is considered to be isolated.Similarly, a nucleic acid or polypeptide sequence may be naturallyexpressed in a cell or organism of a member of the halomicrobium genus,but the sequence is not part of or included in a halomicrobium cell ororganism, it is considered to be isolated. Thus, a nucleic acid orpolypeptide sequence of a haloarcula, halomicrobium, or otherhalorhodopsin that is present in a vector, in a heterologous cell,tissue, or organism, etc., is an isolated sequence. The term“heterologous” as used herein, means a cell, tissue or organism that isnot the native cell, tissue, or organism. The terms, “protein”,“polypeptides”, and “peptides” are used interchangeably herein.

LAIP Sequences Including Modified Sequences

A LAIP of the invention may comprise a wild-type polypeptide sequence ormay be a modified polypeptide sequence. As used herein the term“modified” or “modification” in reference to a nucleic acid orpolypeptide sequence refers to a change of one, two, three, four, five,six, or more amino acids in the sequence as compared to the wild-typesequence from which it was derived. For example, a modified polypeptidesequence may be identical to a wild-type polypeptide sequence exceptthat it has one, two, three, four, five, or more amino acidsubstitutions, deletions, insertions, or combinations thereof. In someembodiments of the invention a modified sequence may include one, two,three, four, or more amino acid substitutions in a wild-typehalorhodopsin sequence.

It will be understood that sequences of LAIPs of the invention may bederived from various members of the haloarcula genus, the halomicrobium,or from other rhodopsin sequences that correspond, at least in part, tohaloarcula and halomicrobium sequences disclosed herein. For example,SEQ ID NO:2, the wild-type amino acid sequence of the halorhodopsinpolypeptide referred to herein as Halo57 is shown in FIG. 3. Amino acidsequences of additional exemplary LAIP polypeptides of the invention arealso shown in FIG. 3, which shows sequences aligned with Halo57 and witheach other. Using standard sequence alignment methods one of ordinaryskill in the art is able to align rhodopsin sequences (including, butnot limited to haloarcula halorhodopsin, halomicrobium halorhodopsin,and other halobacteriaceae halorhodopsin sequences) to determine thecorrespondence of a residue in one sequence with a residue in an alignedsequence. Thus, as a non-limiting example, one skilled in the art canascertain that the Ala residue at position 104 in the Gene56 sequenceset forth as SEQ ID NO:9; corresponds to the Ala residue at position 88in the Gene55 sequence set forth as SEQ ID NO:10 and that bothcorrespond to the Ala residue at position 122 in the Halo57 sequence setforth as SEQ ID NO:2.

Routine sequence alignment methods and techniques can be used to aligntwo or more substantially similar rhodopsin sequences, including but notlimited to sequences from haloarcula rhodopsin, halomicrobiumhalorhodopsin, etc., thus providing a means by which a correspondinglocation of a modification made in one LAIP sequence can be identifiedin another rhodopsin sequence. For example, the correspondingposition(s) of modifications such as A122D, K200R, W214F in the Halo57sequence set forth as SEQ ID NO:2 can be identified in alignedsequences. Similarly, the corresponding position(s) of modificationssuch as A137D, K215R, W229F in the Halo sequence set forth as SEQ IDNO:14 can be identified in aligned sequences. The substituted Halo57sequence can be aligned with one or more haloarcula halorhodopsinsequences or with one or more other rhodopsin sequence that aresubstantially similar in amino acid sequence to Halo57 as set forthherein, to identify corresponding positions for the substitutions in thealigned sequences. LAIP polypeptides having one or more substitutions orother modifications can be identified and tested for characteristicsincluding, but not limited to: expression, cell localization, activationand silencing in response to contact with light using methods disclosedherein.

A LAIP polypeptide of the invention may include amino acid variants(e.g., polypeptides having a modified sequence) of the naturallyoccurring wild-type sequences, as set forth herein. Modified LAIPpolypeptide sequences may be at least about 75%, 80%, 85%, 90%, 95%,96%, 97%, 98%, 99%, or 100% homologous to the polypeptide sequence of aLAIP disclosed herein, such as Halo57, Gene4, Gene58, etc. Homology inthis context means sequence similarity or identity. Such sequencehomology can be determined using standard techniques known in the art.LAIPs of the present invention include the LAIP polypeptide and nucleicacid sequences provided herein and variants that are more than about50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, or 98%homologous to a provided sequence.

LAIPs polypeptide of the invention may be shorter or longer than theLAIP polypeptide sequences set forth herein. Thus, in a preferredembodiment, included within the definition of LAIP polypeptides arefull-length polypeptides or functional fragments thereof. In addition,nucleic acids of the invention may be used to obtain additional codingregions, and thus additional protein sequences, using techniques knownin the art.

In some aspects of the invention, substantially similar Halobacteriaceaehalorhodopsin polypeptide sequences may have at least 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or 100% similarity to ahalorhodopsin sequence disclosed herein, non-limiting examples of whichinclude as Halo57, Gene58, Gene4, and Gene 56, etc. Art-known alignmentmethods and tools can be used to align substantially similar sequencespermitting positional identification of amino acids that may be modifiedas described herein to prepare a LAIP of the invention.

Sequence modifications can be in one or more of three classes:substitutions, insertions or deletions. These modified sequences, (whichmay also be referred to as variants) ordinarily are prepared by sitespecific mutagenesis of nucleic acids in the DNA encoding a LAIPpolypeptide, using cassette or PCR mutagenesis or other techniques knownin the art, to produce DNA encoding the modified LAIP, and thereafterexpressing the DNA in recombinant cell culture. Amino acid sequencevariants are characterized by the predetermined nature of the variation,a feature that sets them apart from naturally occurring allelic orinterspecies variation of the LAIPs of the invention. Modified LAIPsgenerally exhibit the same qualitative biological activity as thenaturally occurring analogue, although variants can also be selectedwhich have modified characteristics.

A site or region for introducing an amino acid sequence modification maybe predetermined, and the mutation per se need not be predetermined. Forexample, to optimize the performance of a mutation at a given site,random mutagenesis may be conducted at the target codon or region andthe expressed modified LAIP screened for the optimal combination ofdesired activity. Techniques for making substitution mutations atpredetermined sites in DNA having a known sequence are well known, forexample, M13 primer mutagenesis and PCR mutagenesis.

Amino acid substitutions are typically of single residues; andinsertions usually will be on the order of from about 1 to 20 aminoacids, although considerably larger insertions may be tolerated.Deletions may range from about 1 to about 20 residues, although in somecases deletions may be much larger.

Substitutions, deletions, insertions or any combination thereof may beused to arrive at a final modified LAIP of the invention. Generallythese changes are done on a few amino acids to minimize the alterationof the molecule. However, larger changes may be tolerated in certaincircumstances.

Variants of LAIPs set forth herein, may exhibit the same qualitativelight-activated ion pump activity as one or more of the sequences setforth herein, such as Halo57, Gene4, Gene57, but may show some alteredcharacteristics such as altered photocurrent, stability, speed,compatibility, and toxicity, or a combination thereof. For example, thepolypeptide can be modified such that it has an increased photocurrentand/or has less toxicity than another LAIP polypeptide.

A LAIP polypeptide of the invention can incorporate unnatural aminoacids as well as natural amino acids. An unnatural amino acid can beincluded in a LAIP of the invention to enhance a characteristic such asphotocurrent, stability, speed, compatibility, or to lower toxicity,etc.

In some embodiments, the invention includes the use of targetedsite-directed mutagenesis at specific amino acid residues ofhalorhodopsins, including but not limited to residues that correspond toA122D, K200R, and W215F of the amino acid sequence set forth as Halo57(SEQ ID NO:2). Specific point mutations have been identified that alone,or in combination of two or more have been demonstrated to beparticularly effective at altering photocurrent amplitude in LAIPs ofthe invention. In particular, the A122D, K200R and W214F mutations tocruxhalorhodopsins and additionally, the Natromonas pharaonishalorhodopsin, have been found boost effective light sensitivity andphotocurrent amplitude to hyperpolarize excitable cells. Substitutions,including but not limited to: K200R, T111S, K200H, K200Q, T203S,[K200Q+W214F], [K200H+W214F], (amino acid numbered in reference to theHalo57 sequence set forth here as SEQ ID NO:2) all were found tosubstantially boost photocurrent. Thus, mutations that correspond to aK200R substitution, a T111S substitution, a K200H substitution, a K200Qsubstitution, a T203S substitution, a K200Q+W214F double substitution,or a K200H+W214F double substitution, can be made in Halo57 sequenceand/or in a Halobacteriaceae halorhodopsin sequence that issubstantially similar to Halo57 to prepare a LAIP of the invention.Substitutions at the K200 position appear to be beneficial and toincrease photocurrent required to hyperpolarize cells. Additionalsubstitutions at the K200 position including, but not limited to: K200Dand K200S (number in reference to the Halo57 amino acid sequenceprovided herein) are also contemplated in embodiments of the invention.Also, substitutions at the W214 position appear to be beneficial and toincrease photocurrent required to hyperpolarize cells, including, butnot limited to a W214Y substitution, a W214L substitution. Additionalsubstitutions at the W214 position (number in reference to the Halo57amino acid sequence provided herein) are also contemplated inembodiments of the invention. The amino acid sequence of Halo57 with thedouble substitution K200R+W214F is set forth as SEQ ID NO:26.

Another aspect of the invention provides nucleic acid sequences thatcode for a LAIP of the invention. It would be understood by a person ofskill in the art that the LAIP polypeptides of the present invention canbe coded for by various nucleic acids. Each amino acid in the protein isrepresented by one or more sets of 3 nucleic acids (codons). Becausemany amino acids are represented by more than one codon, there is not aunique nucleic acid sequence that codes for a given protein. It is wellunderstood by those of skill in the art how to make a nucleic acid thatcan code for LAIP polypeptides of the invention by knowing the aminoacid sequence of the protein. A nucleic acid sequence that codes for apolypeptide or protein is the “gene” of that polypeptide or protein. Agene can be RNA, DNA, or other nucleic acid than will code for thepolypeptide or protein.

It is understood in the art that the codon systems in differentorganisms can be slightly different, and that therefore where theexpression of a given protein from a given organism is desired, thenucleic acid sequence can be modified for expression within thatorganism. Thus, in some embodiments, a LAIP polypeptide of the inventionis encoded by a mammalian-codon-optimized nucleic acid sequence, whichmay in some embodiments be a human-codon optimized nucleic acidsequence. An aspect of the invention provides a nucleic acid sequencethat codes for a LAIP that is optimized for expression with a mammaliancell. A preferred embodiment comprises a nucleic acid sequence optimizedfor expression in a human cell.

Delivery of LAIPs

Delivery of a LAIP polypeptide to a cell and/or expression of a LAIP ina cell can be done using art-known delivery means.

In some embodiments of the invention a LAIP polypeptide of the inventionis included in a fusion protein. It is well known in the art how toprepare and utilize fusion proteins that comprise a polypeptidesequence. In certain embodiments of the invention, a fusion protein canbe used to deliver a LAIP to a cell and can also in some embodiments beused to target a LAIP of the invention to specific cells or to specificcells, tissues, or regions in a subject. Targeting and suitabletargeting sequences for deliver to a desired cell, tissue or region canbe performed using art-known procedures.

It is an aspect of the invention to provide a LAIP polypeptide of theinvention that is non-toxic, or substantially non-toxic in cells inwhich it is expressed. In the absence of light, a LAIP of the inventiondoes not significantly alter cell health or ongoing electrical activityin the cell in which it is expressed.

In some embodiments of the invention, a LAIP of the invention isgenetically introduced into a cellular membrane, and reagents andmethods are provided for genetically targeted expression of LAIPpolypeptides, including Halo57, Gene4, Gene58, etc. Genetic targetingcan be used to deliver LAIP polypeptides to specific cell types, tospecific cell subtypes, to specific spatial regions within an organism,and to sub-cellular regions within a cell. Genetic targeting alsorelates to the control of the amount of LAIP polypeptide expressed, andthe timing of the expression.

Some embodiments of the invention include a reagent for geneticallytargeted expression of a LAIP polypeptide, wherein the reagent comprisesa vector that contains the gene for the LAIP polypeptide.

As used herein, the term “vector” refers to a nucleic acid moleculecapable of transporting between different genetic environments anothernucleic acid to which it has been operatively linked. The term “vector”also refers to a virus or organism that is capable of transporting thenucleic acid molecule. One type of vector is an episome, i.e., a nucleicacid molecule capable of extra-chromosomal replication. Some usefulvectors are those capable of autonomous replication and/or expression ofnucleic acids to which they are linked. Vectors capable of directing theexpression of genes to which they are operatively linked are referred toherein as “expression vectors”. Other useful vectors, include, but arenot limited to viruses such as lentiviruses, retroviruses, adenoviruses,and phages. Vectors useful in some methods of the invention cangenetically insert LAIP polypeptides into dividing and non-dividingcells and can insert LAIP polypeptides to cells that are in vivo, invitro, or ex vivo cells.

Vectors useful in methods of the invention may include additionalsequences including, but not limited to one or more signal sequencesand/or promoter sequences, or a combination thereof. Expression vectorsand methods of their use are well known in the art. Non-limitingexamples of suitable expression vectors and methods for their use areprovided herein.

In certain embodiments of the invention, a vector may be a lentiviruscomprising the gene for a LAIP of the invention, such as Halo57, Gene4,Gene58, or a variant thereof. A lentivirus is a non-limiting example ofa vector that may be used to create stable cell line. The term “cellline” as used herein is an established cell culture that will continueto proliferate given the appropriate medium.

Promoters that may be used in methods and vectors of the inventioninclude, but are not limited to, cell-specific promoters or generalpromoters. Methods for selecting and using cell-specific promoters andgeneral promoters are well known in the art. A non-limiting example of ageneral purpose promoter that allows expression of a LAIP polypeptide ina wide variety of cell types—thus a promoter for a gene that is widelyexpressed in a variety of cell types, for example a “housekeeping gene”can be used to express a LAIP polypeptide in a variety of cell types.Non-limiting examples of general promoters are provided elsewhere hereinand suitable alternative promoters are well known in the art.

In certain embodiments of the invention, a promoter may be an induciblepromoter, examples of which include, but are not limited totetracycline-on or tetracycline-off, or tamoxifen-inducible Cre-ER.

Methods of Use of LAIPs of the Invention

LAIPs of the invention are well suited for targeting hemoglobin-richtissues, such as the heart, cardiac system, arterial walls, or highcapillary density due to the fact that they have unique activationspectra that prevents optical attenuation from oxygenated hemoglobin orpure hemoglobin absorption. FIG. 4A illustrates a trough of hemoglobinabsorption and shows results indicating that oxygen-bound hemoglobinabsorbs 24 times more light at 593 nm illumination than at 660 nm, nearits trough of absorbance. FIG. 4B shows results indicating that Halo57redshift, which allows illumination at the hemoglobin absorption trough.

Further, their low levels of hyperpolarization and lack of silencing atblue light wavelengths is well suited for optogenetic applications insubjects or patients where blue-light illumination is unavoidable, suchas photodynamic “blue-light” therapy for actinic keratosis and acnepatients, or the blue light boxes used to induce melatonin productionfor sufferers of seasonal affective disorder (SAD).

Working operation of a prototype of this invention was demonstrated bygenetically expressing light-activated ion pump molecules of theinvention in excitable cells, illuminating the cells with suitablewavelengths of light, and demonstrating rapid hyperpolarization of thecells in response to the light, as well as rapid release fromhyperpolarization upon cessation of light. Depending on the particularimplementation, methods of the invention allow light control of cellularfunctions in vivo, ex vivo, and in vitro.

In some aspects of the invention, the newly identified light-activatedion pumps are chloride pumps, which can be used to modify thetransmembrane potential of cells (and/or their sub-cellular regions, andtheir local environment). For example, the use of inwardly rectifyingchloride pumps will hyperpolarize cells by moving negatively chargedchloride ions from the extracellular environment into the cytoplasm, andthe red light sensitivity of light-activated ion pumps of the invention,with respect to cell silencing, is highly advantageous due to the lowlight power requirement, as well as the fact that the pumps can be usedin conjunction with other optically sensitive molecules with differentaction spectra to control multiple cell types with reduced interferencefrom cross-excitation.

In non-limiting examples of methods of the invention, microbialrhodopsins are used in mammalian cells without need for any kind ofchemical supplement, and in normal cellular environmental conditions andionic concentrations. For example, members of the haloarcula class, suchas the Halobacterium salinarum (strain shark)/Halobacterium halobium(strain shark) gene for halorhodopsin, the Halobacterium salinarum(strain port)/Halobacterium halobium (strain port) gene forhalorhodopsin, the Haloarcula marismortui ATCC 43049 gene forcruxhalorhodopsin, the Halomicrobium mukohataei DSM 12286 gene forcruxhalorhodopsin, the Haloarcula californiae ATCC 33799 gene forcruxhalorhodopsin, and the Haloarcula sinaiiensis ATCC 33800 gene forcruxhalorhodopsin have been used in exemplary implementations of theinvention. The polypeptides encoded by these genes, e.g., in humanizedor mouse-optimized form, allow hyperpolarizations significantly largerthan what has been discovered before at red-light wavelengths.

In exemplary implementations of this invention, the Halobacteriumsalinarum (strain shark)/Halobacterium halobium (strain shark) gene forhalorhodopsin, the Halobacterium salinarum (strain port)/Halobacteriumhalobium (strain port) gene for halorhodopsin, and the Haloarculamarismortui ATCC 43049 gene for cruxhalorhodopsin have demonstrablyimproved photocurrent generation at red wavelengths, with significantlylower power requirements. For example, the Halobacterium salinarum(strain shark)/Halobacterium halobium (strain shark) gene forhalorhodopsin (also referred to herein as Halo57) produces 2.9× morephotocurrent than the gene product (N. pharaonis halorhodopsin, alsodenoted as “NpHR” or “Halo”) at 4 mW/mm² yellow light illumination and44× the photocurrent at 7 mW/mm² red light illumination (see FIG. 5A),respectively (yellow light wavelength=575±25 nm, red lightwavelength=660 nm. FIG. 5B shows that photocurrents for Halo57 can befurther improved by inclusion of a K200R+W214F double mutation.

As used herein, the term “ion pump” means an integral membranepolypeptide that is capable of moving ions across the membrane of acell. In general, an ion pump comprises one or more proteins located ina cell membrane. Ion pumps may actively transport ions across themembrane against a concentration gradient using ATP. General functionsof ion pumps may include, but are not limited to maintaining osmoticbalance in a cell or conducting nerve impulses. Many ion pumps do notexpress well in a cell and/or their expression may be toxic to the celland reduce cell health. Thus it was necessary to prepare and screennumerous halorhodopsin light-activated ion pump polypeptides includingnumerous pumps having one, two, or more specific mutations to identifylight-activated ion pumps of the invention that can be expressed incells without significantly reducing cell health and viability.

Light-activated ion pumps of the invention have been found to besuitable for expression and use in mammalian cells without need for anykind of chemical supplement, and in normal cellular environmentalconditions and ionic concentrations. LAIPs of the invention have beenfound to differ from previously identified pumps in that the LAIPsactivate at a wavelengths of light ranging from about 450 nm to about690 nm, but they also are specifically sensitive to red light and a cellthat expresses a LAIP of the invention will be silenced with the LAIP ofthe invention is contacted with a red light.

Cells and Subjects

A cell used in methods and with sequences of the invention may be anexcitable cell or a non-excitable cell. A cell in which a LAIP of theinvention may be expressed and may be used in methods of the inventioninclude prokaryotic and eukaryotic cells. Useful cells include but arenot limited to mammalian cells. Examples of cells in which a LAIP of theinvention may be expressed are excitable cells, which include cells ableto produce and respond to electrical signals. Examples of excitable celltypes include, but are not limited to neurons, muscles, cardiac cells,and secretory cells (such as pancreatic cells, adrenal medulla cells,pituitary cells, etc.). LAIPs of the invention may also be expressed innon-excitable cells and may function therein, when activated by redlight, to alter the ion conductance in the non-excitable cells. Forexample, an LAIP of the invention may permit chloride transport mayacross a membrane of a non-excitable cell. LAIPs and methods of theinvention may include use of excitable or non-excitable cells in methodsof candidate compound assessment, diagnosis, and treatment. Examples ofnon-excitable cells to which LAIPs of the invention and methods usingLAIP of the invention include, but are not limited to cells of the lung,pancreas, liver, intestine, skin, etc.

Non-limiting examples of cells that may be used in methods of theinvention include nervous system cells, cardiac cells, circulatorysystem cells, visual system cells, auditory system cells,hemoglobin-rich cells, secretory cells, endocrine cells, or musclecells. In some embodiments, a cell used in conjunction with theinvention may be a healthy normal cell, which is not known to have adisease, disorder or abnormal condition. In some embodiments, a cellused in conjunction with methods and pumps of the invention may be anabnormal cell, for example, a cell that has been diagnosed as having adisorder, disease, or condition, including, but not limited to adegenerative cell, a neurological disease-bearing cell, a cell model ofa disease or condition (e.g., cystic fibrosis, blindness, hearing loss,cardiac disease, etc.), an injured cell, etc. In some embodiments of theinvention, a cell may be a control cell.

LAIPs of the invention may be expressed in cells from culture, cells insolution, cells obtained from subjects, and/or cells in a subject (invivo cells). LAIPs may be expressed and activated in cultured cells,cultured tissues (e.g., brain slice preparations, etc.), and in livingsubjects, etc. As used herein, a the term “subject” may refer to ahuman, non-human primate, cow, horse, pig, sheep, goat, dog, cat,rodent, fly or any other vertebrate or invertebrate organism.

Controls and Candidate Compound Testing

LAIPs of the invention and methods using LAIPs of the invention can beutilized to assess changes in cells, tissues, and subjects in which theyare expressed. Some embodiments of the invention include use of LAIPs ofthe invention to identify effects of candidate compounds on cells,tissues, and subjects. Results of testing a LAIP of the invention can beadvantageously compared to a control.

As used herein a control may be a predetermined value, which can take avariety of forms. It can be a single cut-off value, such as a median ormean. It can be established based upon comparative groups, such as cellsor tissues that include the LAIP and are contacted with light, but arenot contacted with the candidate compound and the same type of cells ortissues that under the same testing condition are contacted with thecandidate compound. Another example of comparative groups may includecells or tissues that have a disorder or condition and groups withoutthe disorder or condition. Another comparative group may be cells from agroup with a family history of a disease or condition and cells from agroup without such a family history. A predetermined value can bearranged, for example, where a tested population is divided equally (orunequally) into groups based on results of testing. Those skilled in theart are able to select appropriate control groups and values for use incomparative methods of the invention.

As a non-limiting example of use of a LAIP to identify a candidatetherapeutic agent or compound, a LAIP of the invention may be expressedin an excitable cell in culture or in a subject and the excitable cellmay be contacted with a light that activates the LAIP and with acandidate therapeutic compound. In one embodiment, a test cell thatincludes a LAIP of the invention can be contacted with a light thathyperpolarizes and/or silences the cell and also contacted with acandidate compound. The cell, tissue, and/or subject that include thecell can be monitored for the presence or absence of a change thatoccurs in the test conditions versus the control conditions. Forexample, in a cell, a change may be a change in the hyperpolarization orin a hyperpolarization-mediated cell characteristic in the test cellversus a control cell, and a change in hyperpolarization or thehyperpolarization-mediated cell characteristic in the test cell comparedto the control may indicate that the candidate compound has an effect onthe test cell or tissue that includes the cell. In some embodiments ofthe invention, a hyperpolarization mediated cell characteristic may be ahyperpolarization-activated conductance, which may, for example, be theresult of a T-type calcium channel activity, BK channel activity, or anI_h current. As known in the art, T-type calcium channels are a type ofhigh-voltage calcium channels, BK channels, also referred to as “bigpotassium” channels are high-conductance potassium channels, and an I_hcurrent is a current that flows through hyperpolarization-activatedcyclic-nucleotide gated (HCN) channels. Means of assessing T-typecalcium channel activity, BK channel activity, I_h currents, and otherhyperpolarization mediated cell characteristics are known in the art. Incertain embodiments, a hyperpolarization-mediated-cell characteristic iscell silencing.

Candidate-compound identification methods of the invention that areperformed in a subject, may include expressing a LAIP in the subject,contacting the subject with a light under suitable conditions toactivate the LAIP and hyperpolarize the cell, and administering to thesubject a candidate compound. The subject is then monitored to determinewhether any change occurs that differs from a control effect in asubject. Thus, for example, a brain region may be silenced using a LAIPof the invention and a candidate compound may be administered to thebrain and the effect of the compound determined by comparing the resultswith those of a control.

Methods of identifying effects of candidate compounds using LAIPs of theinvention may also include additional steps and assays to furthercharacterizing an identified change in the cell, tissue, or subject whenthe cell is contacted with the candidate compound. In some embodiments,testing in a cell, tissue, or subject can also include one or more cellsthat has a LAIP of the invention, and that also has one, two, three, ormore additional different light-activated ion pumps, wherein at leastone, two, three, four, or more of the additional light-activated ionpumps is activated by contact with light having a non-red lightwavelength.

In a non-limiting example of a candidate drug identification method ofthe invention, cells that include a LAIP of the invention arehyperpolarized, thus activating endogenous hyperpolarization-activatedconductances (such as T-type calcium channels, BK channels, and I_hcurrents), and then drugs are applied that modulate the response of thecell to hyperpolarization (determined for example using patch clampingmethods or other suitable art-known means). Such methods enable newkinds of drug screening using just light to activate the pumps ofinterest, and using just light to read out the effects of a drug on thepumps and pump-containing cells of interest.

In some embodiments, LAIP polypeptides of the invention can be used intest systems and assays for assessing membrane protein trafficking andphysiological function in heterologously expressed systems and the useof use of light-activated pumps to hyperpolarize a cell that has highintracellular chloride concentrations, such as young adult-born neurons.LAIPs of the invention can also be used test compounds to treat diseasesor conditions such as cystic fibrosis, blindness, pain, seizures,degenerative disease, developmental disease, etc.

Methods of Treating

Some aspects of the invention include methods of treating a disorder orcondition in a cell, tissue, or subject using LAIPs of the invention.Treatment methods of the invention may include administering to asubject in need of such treatment, a therapeutically effective amount ofa LAIP of the invention to treat the disorder. It will be understoodthat a treatment may be a prophylactic treatment or may be a treatmentadministered following the diagnosis of a disease or condition. Atreatment of the invention may reduce or eliminate a symptom orcharacteristic of a disorder, disease, or condition or may eliminate thedisorder, disease, or condition itself. It will be understood that atreatment of the invention may reduce or eliminate progression of adisease, disorder or condition and may in some instances result in theregression of the disease, disorder, or condition. A treatment need toentirely eliminate the disease, disorder, or condition to be effective.

Administration of a LAIP of the invention may include administrationpharmaceutical composition that includes a cell, wherein the cellexpresses the light-activated ion pump. Administration of a LAIP of theinvention may include administration of a pharmaceutical compositionthat includes a vector, wherein the vector comprises a nucleic acidsequence encoding the light-activated ion pump and the administration ofthe vector results in expression of the light-activated ion pump in acell in the subject.

An effective amount of a LAIP is an amount that increases LAIP in acell, tissue or subject to a level that is beneficial for the subject.An effective amount may also be determined by assessing physiologicaleffects of administration on a cell or subject, such as a decrease insymptoms following administration. Other assays will be known to one ofordinary skill in the art and can be employed for measuring the level ofthe response to a treatment. The amount of a treatment may be varied forexample by increasing or decreasing the amount of the LAIP administered,by changing the therapeutic composition in which the LAIP isadministered, by changing the route of administration, by changing thedosage timing and so on. The effective amount will vary with theparticular condition being treated, the age and physical condition ofthe subject being treated; the severity of the condition, the durationof the treatment, the nature of the concurrent therapy (if any), thespecific route of administration, and the like factors within theknowledge and expertise of the health practitioner. For example, aneffective amount may depend upon the location and number of cells in thesubject in which the LAIP is to be expressed. An effective amount mayalso depend on the location of the tissue to be treated.

Effective amounts will also depend, of course, on the particularcondition being treated, the severity of the condition, the individualpatient parameters including age, physical condition, size and weight,the duration of the treatment, the nature of concurrent therapy (ifany), the specific route of administration and like factors within theknowledge and expertise of the health practitioner. These factors arewell known to those of ordinary skill in the art and can be addressedwith no more than routine experimentation. It is generally preferredthat a maximum dose of a composition to increase the level of a LAIP(alone or in combination with other therapeutic agents) be used, thatis, the highest safe dose according to sound medical judgment. It willbe understood by those of ordinary skill in the art, however, that apatient may insist upon a lower dose or tolerable dose for medicalreasons, psychological reasons or for virtually any other reasons.

A LAIP of the invention may be administered using art-known methods. Themanner and dosage administered may be adjusted by the individualphysician or veterinarian, particularly in the event of anycomplication. The absolute amount administered will depend upon avariety of factors, including the material selected for administration,whether the administration is in single or multiple doses, andindividual subject parameters including age, physical condition, size,weight, and the stage of the disease or condition. These factors arewell known to those of ordinary skill in the art and can be addressedwith no more than routine experimentation.

Pharmaceutical compositions that deliver LAIPs of the invention may beadministered alone, in combination with each other, and/or incombination with other drug therapies, or other treatment regimens thatare administered to subjects. A pharmaceutical composition used in theforegoing methods preferably contain an effective amount of atherapeutic compound that will increase the level of a LAIP polypeptideto a level that produces the desired response in a unit of weight orvolume suitable for administration to a subject.

The dose of a pharmaceutical composition that is administered to asubject to increase the level of LAIP in cells of the subject can bechosen in accordance with different parameters, in particular inaccordance with the mode of administration used and the state of thesubject. Other factors include the desired period of treatment. In theevent that a response in a subject is insufficient at the initial dosesapplied, higher doses (or effectively higher doses by a different, morelocalized delivery route) may be employed to the extent that patienttolerance permits. The amount and timing of activation of a LAIP of theinvention (e.g., light wavelength, length of light contact, etc.) thathas been administered to a subject can also be adjusted based onefficacy of the treatment in a particular subject. Parameters forillumination and activation of LAIPs that have been administered to asubject can be determined using art-known methods and without requiringundue experimentation.

Various modes of administration will be known to one of ordinary skillin the art that can be used to effectively deliver a pharmaceuticalcomposition to increase the level of LAIP in a desired cell, tissue orbody region of a subject. Methods for administering such a compositionor other pharmaceutical compound of the invention may be topical,intravenous, oral, intracavity, intrathecal, intrasynovial, buccal,sublingual, intranasal, transdermal, intravitreal, subcutaneous,intramuscular and intradermal administration. The invention is notlimited by the particular modes of administration disclosed herein.Standard references in the art (e.g., Remington's PharmaceuticalSciences, 18th edition, 1990) provide modes of administration andformulations for delivery of various pharmaceutical preparations andformulations in pharmaceutical carriers. Other protocols which areuseful for the administration of a therapeutic compound of the inventionwill be known to one of ordinary skill in the art, in which the doseamount, schedule of administration, sites of administration, mode ofadministration (e.g., intra-organ) and the like vary from thosepresented herein.

Administration of a cell or vector to increase LAIP levels in a mammalother than a human; and administration and use of LAIPs of theinvention, e.g. for testing purposes or veterinary therapeutic purposes,is carried out under substantially the same conditions as describedabove. It will be understood by one of ordinary skill in the art thatthis invention is applicable to both human and animals. Thus thisinvention is intended to be used in husbandry and veterinary medicine aswell as in human therapeutics.

In some aspects of the invention, methods of treatment using a LAIP ofthe invention are applied to cells including but not limited to anervous system cell, a neuron, a cardiac cell, a circulatory systemcell, a visual system cell, an auditory system cell, a hemoglobin-richcell, a muscle cell, or an endocrine cell. Disorders and conditions thatmay be treated using methods of the invention include, injury, braindamage, degenerative neurological conditions, and may include treatmentof diseases and conditions such as Parkinson's disease, Alzheimer'sdisease, seizure, vision loss, (e.g., retinitis pigmentosa, etc.),hearing loss, cystic fibrosis, pain, etc. Methods of treatment thatutilize light activated pumps and channels are known in the art. See,for example. Busskamp, V. et al., Science. 2010 Jul. 23;329(5990):413-7. Epub 2010 Jun. 24, incorporated herein by reference.

Disorders, Diseases, and Conditions

In some aspects of the invention, hemoglobin rich cell types and tissuesare targeted using LAIPs of the invention. These comprise the heart,liver, erythrocytes, kidneys, blood vessel walls, or other tissue typeswith a high surrounding concentration of arteries, veins, orcapillaries. Such targeting may be used to advantage for medicaltreatments, since a high concentration of blood vessels often rendersthese regions difficult to surgically access. Additionally, theoptogenetic control of cardiac and circulatory tissues may be used as anon-pharmacological alternative for the optical control of bloodpressure through the vasodilation and vasoconstriction of arteries andveins.

In certain embodiments, methods and LAIPs of the invention may be usedto silence large tissue volumes or whole-brain optical silencing as analternative to methods such as deep brain stimulation (DBS). In someembodiments, methods and LAIPs of the invention may be used to targettissues which are physically difficult to non-destructively access, suchas regions of the brain.

In some embodiments, methods and LAIPs of the invention may be used forthe long-term treatment for neural disorders with overactive neuralbehavior, such as epilepsy or muscular spasticity. The low light powerrequirement means it is well-suited for repeated, long term use sincethe cumulative effect of surrounding tissue illumination and irradiancewould be dramatically lessened.

In some embodiments methods and LAIPs of the invention may be used forneural silencing, as an alternative for blue-light sensitive opsins suchas Mac and Halo, where blue-light illumination might cause undesirableopsin behavior. This alternative is particularly advantageous forpatients who undergo blue light illumination for another condition, suchas the photodynamic “blue-light” therapy used to treat acne and actinickeratosis, or the blue light boxes which are used to address seasonalaffective disorder (SAD).

In some embodiments, methods and LAIPs of the invention may be used forthe treatment of inner ear hearing or balance disorders. Its low lightpower requirement for optical stimulation is of great use for cochlearimplant alternatives, since stray heat from light irradiance will causehair cells to fire. Additionally, current cochlear implants areproblematic due to the fact that the operation required to access thecochlear hair cells is physically destructive and destroys the patient'sresidual hearing. The large tissue volume silencing properties which theLAIPs possess allow a minimally invasive stimulation of the hair cells,and reduce the destruction of auditory tissue.

In some embodiments, methods and LAIPs of the invention may be used forthe treatment of visual system disorders, for example to treat visionreduction or loss. A LAIP of the invention may be administered to asubject who has a vision reduction or loss and the expressed LAIPs canfunction as light-sensitive cells in the visual system, therebypermitting a gain of visual function in the subject.

Exemplary treatment methods of the invention may also include use ofLAIPs for deep brain silencers or deep brain inhibitors (hereby termedDBSi or DBI, respectively) in the mammalian brain. Deep brain silencingmay be used in conjunction with deep brain stimulation, for example, tolimit adverse side effects created by electrical stimulation thataffects all cell types.

LAIPs of the invention are useful in methods, including, but not limitedto prosthetic applications such as gene therapy+device applications inwhich excitable cells (heart cells, neuron, muscle cells, endocrinecells, etc.) can be silenced, in order to produce long-term cellsilencing for neural prosthetics and treatments of disease, which arewell suited for the treatment of epilepsy, Parkinson's, neuromuscularconditions and other disorders; drug screening applications including,but not limited to, hyperpolarizing cells, thus activating endogenoushyperpolarization-activated conductances (such as T-type calciumchannels, BK channels, and I_h currents), and then applying drugs thatmodulate the response of the cell to hyperpolarization (using a calciumor voltage-sensitive dye); diagnostics applications, such as, but notlimited to, sensitizing samples of tissues from patients to light, orconverting them into other cell types (e.g., stem cells) and thensensitizing those to light; and the collection of optical energy, e.g.solar energy, using light-activated pumps expressed in cell lines.

The present invention in some aspects, includes preparing nucleic acidsequences and polynucleotide sequences; expressing in excitable cellspolypeptides encoded by the prepared nucleic acid and polynucleotidesequences; illuminating the cells with suitable light, and demonstratingrapid hyperpolarization of the cells in response to light, as well asrapid release from hyperpolarization upon cessation of light. Theability to controllably alter hyperpolarization with light has beendemonstrated. The present invention enables light-control of cellularfunctions in vivo, ex vivo, and in vitro, and the red-light activatedion pumps of the invention and their use, have broad-rangingapplications for drug screening, treatments, and research applications,some of which are describe herein.

In some instantiations of this invention, sensitizing chromophores (suchas chlorophyll or salinixanthin) are used to broaden or shift theabsorbance spectrum of the molecule, and are particularly advantageousfor multi-color silencing, tuning the absorbance for optimality withspecific optical apparatus (e.g. narrow excitation LEDs and lasers, longwavelength absorption for better transmission through tissue, etc.), orthe creation of harmful UV oxidized species.

This invention may be used to advantage to enhance the functionalperformance of the heterologously expressed ion pumps in mammalian cellsvia site-directed mutagenesis, such as the A122D single mutation of Haloand the K200R+W214F double mutant of the Halobacterium salinarum (strainshark)/Halobacterium halobium (strain shark) gene for halorhodopsin.

In illustrative implementations of this invention, the ability tooptically perturb, modify, or control cellular function offers manyadvantages over physical manipulation mechanisms. These advantagescomprise speed, non-invasiveness, and the ability to easily span vastspatial scales from the nanoscale to macroscale.

The reagents use in the present invention (and the class of moleculesthat they represent), allow, at least: significantly larger currentsthan any previous reagent under red-light illumination; differentspectra from older molecules (opening up multi-color control of cells);and greater light sensitivity and tissue penetration depth for opticalsilencing, making it possible to silence much larger tissue volumes, orsilence a target region at a significantly greater distance from theoptical stimulus.

EXAMPLES Example 1

Studies were performed to prepare sequences and to expresslight-activated ion pumps in cells, tissues, and subjects. Non-limitingexemplary methods are set forth below. Art-known methods that may beapplied to light-activated pump molecules and for their use aredisclosed in publications such as US Published Application No.2010/0234273, US Published Application No. 2011/0165681, Chow B Y, et.al. Methods Enzymol. 2011; 497:425-43; Chow, B Y, et al. Nature 2010Jan. 7; 463(7277):98-102, the content of each of which is incorporatedby reference herein.

Plasmid Construction and Site Directed Mutagenesis.

Opsins were mammalian codon-optimized, and synthesized by Genscript(Genscript Corp., NJ). Opsins were fused in frame, without stop codons,ahead of GFP (using BamHI and AgeI) in a lentiviral vector containingthe CaMKII promoter, enabling direct neuron transfection, HEK celltransfection (expression in HEK cells is enabled by a ubiquitouspromoter upstream of the lentiviral cassette), and lentivirus productionand transfection.

Amino acid sequences of various opsins are shown in FIG. 3.

The ‘ss’ signal sequence from truncated MHC class I antigen correspondedto amino acid sequence (M)VPCTLLLLLAAALAPTQTRA (SEQ ID NO:16), DNAsequence gtcccgtgcacgctgctcctgctgttggcagccgccctggctccgactcagacgcgggcc(SEQ ID NO:15). The ‘Prl’ Prolactin signal sequence corresponded toamino acid sequence MDSKGSSQKGSRLLLLLVVSNLLLCQVVS (SEQ ID NO:18), or maybe the amino acid sequence DSKGSSQKGSRLLLLLVVSNLLLCQGVVSTPV (SEQ IDNO:27); DNA sequencegacagcaaaggttcgtcgcagaaagggtcccgcctgctcctgctgctggtggtgtcaaatctactcttgtgccagggtgtggtctccacccccgtc (SEQ ID NO:17). The ‘ER2’ ER export sequence corresponded toamino acid sequence FCYENEV (SEQ ID NO:20), DNA sequencettctgctacgagaatgaagtg (SEQ ID NO:19). The “KGC-ER2” sequencecorresponded to amino acid sequence KSRITSEGEYIPLDQIDINV and FCYENEV(SEQ ID NO:22 and SEQ ID NO:20, respectively); DNA sequences set forthas SEQ ID NO:21 and SEQ ID NO:19, respectively).

Halo point mutants A137D, K215R, W229F, A137D+K215R, A137D+W229F,K215R+W229F, A137D+K215R+W229F) numbered in reference to the Halosequence set forth herein as SEQ ID NO:14), for HEK cell testing weregenerated using the QuikChange kit (Stratagene) on the Halo-GFP fusiongene, corresponding to amino acid sequence set forth herein as SEQ IDNO: 24 or SEQ ID NO:25 and having DNA sequence set forth herein as SEQID NO:23 inserted between BamHI and EcoRI sites in the pcDNA3.1 backbone[Invitrogen, (Life Technologies Corporation, Carlsbad, Calif.)]. Allother point mutants for HEK cell testing were generated using theQuikChange kit [Stratagene, (Agilent Technologies, Santa Clara, Calif.)]on the opsin-GFP fusion gene inserted between BamHI and AgeI sites in amodified version of the pEGFP-N3 backbone [Invitrogen, (LifeTechnologies Corporation, Carlsbad, Calif.)]. All constructs wereverified by sequencing. Note that mutations in the correspondingpositions in the Halo57 sequence set forth herein as SEQ ID NO:2 are asfollows: A122D; K200R; W214F, A122D+K200R; A122D+W214F; K200R+W214F;A122D+K200R+W214F, and such mutants were prepared and tested, in Halo57and in other sequences set forth herein, such as for example, Gene4,Gene55, Gene56, etc., at their corresponding amino acid positions.

Neuron Culture, Transfection, Infection, and Imaging

All procedures involving animals were in accordance with the NationalInstitutes of Health Guide for the care and use of laboratory animalsand approved by the Massachusetts Institute of Technology Animal Careand Use Committee. Swiss Webster or C57 mice (Taconic, Hudson, N.Y. orThe Jackson Laboratory, Bar Harbor, Mass.) were used. For hippocampalcultures, hippocampal regions of postnatal day 0 or day 1 mice wereisolated and digested with trypsin (1 mg/ml) for ˜12 min, and thentreated with Hanks solution supplemented with 10-20% fetal bovine serumand trypsin inhibitor (Sigma-Aldrich, St. Louis, Mo.). Tissue was thenmechanically dissociated with Pasteur pipettes, and centrifuged at 1000rpm at 4° C. for 10 min. Dissociated neurons were plated at a density ofapproximately four hippocampi per 20 glass coverslips, coated withMatrigel (BD Biosciences, Sparks, Md.). For cortical cultures,dissociated mouse cortical neurons (postnatal day 0 or 1) were preparedas previously described, and plated at a density of 100-200 k per glasscoverslip coated with Matrigel (BD Biosciences, Sparks, Md.). Cultureswere maintained in Neurobasal Medium supplemented with B27 [Invitrogen,(Life Technologies Corporation, Carlsbad, Calif.)] and glutamine.Hippocampal and cortical cultures were used interchangeably; nodifferences in reagent performance were noted.

Neurons were transfected at 3-5 days in vitro using calcium phosphate[Invitrogen, (Life Technologies Corporation, Carlsbad, Calif.)]. GFPfluorescence was used to identify successfully transfected neurons.Alternatively, neurons were infected with 0.1-3 μl of lentivirus oradeno-associated virus (AAV) per well at 3-5 days in vitro.

HEK 293FT Cell Culture and Transfection

HEK 293FT cells [Invitrogen, (Life Technologies Corporation, Carlsbad,Calif.)] were maintained between 10-70% confluence in D10 medium(Cellgro, Manassas, Va.) supplemented with 10% fetal bovine serum[Invitrogen, (Life Technologies Corporation, Carlsbad, Calif.)], 1%penicillin/streptomycin (Cellgro, Manassas, Va.), and 1% sodium pyruvate(Biowhittaker, Walkersville, Md.)). For recording, cells were plated at5-20% confluence on glass coverslips coated with Matrigel (BDBiosciences, Sparks, Md.). Adherent cells were transfected approximately24 hours post-plating either with TransLT 293 lipofectamine transfectionkits (Minis, Madison, Wis.) or with calcium phosphate transfection kits[Invitrogen, (Life Technologies Corporation, Carlsbad, Calif.)], andrecorded via whole-cell patch clamp between 36-72 hourspost-transfection.

Lentivirus Preparation

HEK293FT cells [Invitrogen, (Life Technologies Corporation, Carlsbad,Calif.)] were transfected with the lentiviral plasmid, the viral helperplasmid pΔ8.74, and the pseudotyping plasmid pMD2.G. The supernatant oftransfected HEK cells containing virus was then collected 48 hours aftertransfection, purified, and then pelleted through ultracentrifugation.Lentivirus pellet was resuspended in phosphate buffered saline (PBS) andstored at −80° C. until further usage in vitro or in vivo. The estimatedfinal titer is approximately 10⁹ infectious units/mL.

Virus Injection in the Adult Mouse.

All procedures were in accordance with the National Institutes of HealthGuide for the Care and Use of Laboratory Animals and approved by theMassachusetts Institute of Technology Committee on Animal Care or theBoston University Institutional Animal Care and Use Committee. Underisoflurane anesthesia, lentivirus or adeno-associated virus (AAV) wasinjected through a craniotomy made in the mouse skull, into the motorcortex (1.75 mm anterior, 1.5 mm lateral, and 1.75 mm deep, relative tobregma. Custom-fabricated plastic headplates were affixed to the skull,and the craniotomy was protected with agar and dental acrylic.

In Vitro Whole Cell Patch Clamp Recording & Optical Stimulation

Whole cell patch clamp recordings were made using a Multiclamp 700Bamplifier, a Digidata 1440 digitizer, and a PC running pClamp (MolecularDevices, Sunnyvale, Calif.). Neurons were bathed in room temperatureTyrode containing 125 mM NaCl, 2 mM KCl, 3 mM CaCl₂, 1 mM MgCl₂, 10 mMHEPES, 30 mM glucose, 0.01 mM NBQX and 0.01 mM GABAzine. The Tyrode pHwas adjusted to 7.3 with NaOH and the osmolarity was adjusted to 300mOsm with sucrose. HEK cells were bathed in a Tyrode bath solutionidentical to that for neurons, but lacking GABAzine and NBQX.Borosilicate glass pipettes (Warner Instruments, Hamden, Conn.) with anouter diameter of 1.2 mm and a wall thickness of 0.255 mm were pulled toa resistance of 3-9 MΩ with a P-97 Flaming/Brown micropipette puller(Sutter Instruments, Novato, Calif.) and filled with a solutioncontaining 125 mM K-gluconate, 8 mM NaCl, 0.1 mM CaCl₂, 0.6 mM MgCl2, 1mM EGTA, 10 mM HEPES, 4 mM Mg-ATP, and 0.4 mM Na-GTP. The pipettesolution pH was adjusted to 7.3 with KOH and the osmolarity was adjustedto 298 mOsm with sucrose. Access resistance was 5-30 MΩ, monitoredthroughout the voltage-clamp recording. Resting membrane potential was˜−60 mV for neurons and ˜−30 mV for HEK 293FT cells in current-clamprecording.

Photocurrents were measured with 500 ms light pulses in neuronsvoltage-clamped at −60 mV, and in HEK 293FT cells voltage-clamped at −30mV. Light-induced membrane hyperpolarizations were measured with 500 mslight pulses in cells current-clamped at their resting membranepotential. Light pulses for all wavelengths except 660 nm and actionspectrum characterization experiments were delivered with a DG-4 opticalswitch with 300 W xenon lamp (Sutter Instruments, Novato, Calif.),controlled via TTL pulses generated through a Digidata signal generator.Green light was delivered with a 575±25 nm bandpass filter (Chroma,Bellows Falls, Vt.) and a 575±7.5 nm bandpass filter (Chroma, BellowsFalls, Vt.). Action spectra were taken with a Till Photonics PolychromeV, 150 W Xenon lamp, 15 nm monochromator bandwidth.

Data was analyzed using Clampfit (Molecular Devices, Sunnyvale, Calif.)and MATLAB (Mathworks, Inc., Natick, Mass.).

In Vivo Rodent Electrophysiology, Optical Stimulation, and DataAnalysis.

Recordings were made in the cortex of headfixed awake mice after virusinjection, using glass microelectrodes of 5-20 MΩ impedance filled withPBS, containing silver/silver-chloride wire electrodes. Signals wereamplified with a Multiclamp 700B amplifier and digitized with a Digidata1440, using pClamp software (Molecular Devices, Sunnyvale, Calif.). A100 mW red laser (SDL-593-050T, Shanghai Dream Laser (Shanghai DreamLasers Technology Co, Ltd, ShangHai, China) was coupled to a 200micron-diameter optical fiber. The laser was controlled via TTL pulsesgenerated through Digidata. Laser light power was measured with an818-SL photodetector (Newport Corporation, Irvine, Calif.). An opticalfiber was attached to the recording glass electrode, with the tip of thefiber ˜600 μm laterally away from and ˜500 μm above the tip of theelectrode (e.g., ˜800 microns from the tip), and guided into the brainwith a Sutter manipulator at a slow rate of ˜1.5 μm/s to minimizedeformation of the cortical surface.

Data was analyzed using MATLAB (Mathworks, Inc., Natick, Mass.). Spikeswere detected and sorted offline using Wave_clus see:vis.caltech.edu/˜rodri/Wave_clus/Wave_clus_home). Neurons suppressedduring light were identified by performing a paired t-test, for eachneuron, between the baseline firing rate during the 5 second periodbefore light onset vs. during the period of 5 second light illumination,across all trials for that neuron, thresholding at the p<0.05significance level. Instantaneous firing rate histograms were computedby averaging the instantaneous firing rate for each neuron, across alltrials, with a histogram time bin of 20 ms duration. To determine thelatency between light onset and the neural response, a 20 ms-longsliding window was swept through the electrophysiology data and theearliest 20 ms period that deviated from baseline firing rate was lookedfor, as assessed by performing a paired t-test for the firing rateduring each window vs. during the baseline period, across all trials foreach neuron. Latency was defined as the time from light onset to thetime at which firing rate was significantly different from baseline forthe following 120 ms. The time for after-light suppression to recoverback to baseline was calculated similarly.

Opsin-fluorophore cassettes were cloned into an AAV backbone usingrestriction sites BamHI/EcoRI or AAV-FLEX backbone using KpnI/BsrGIsites. The plasmids were amplified and sent to the University of NorthCarolina Chapel Hill Virus Core facility for viral production.

Example 2 Mutation Preparation and Functional Assessment Methods

Using methods set forth in Example 1, a K→R substitution was made at theposition corresponding to amino acid 200 of Halo57 (SEQ ID NO:2) and thesubstituted sequence expressed and tested in a cell.

Using methods set forth in Example 1, a W→F substitution was made at theposition corresponding to amino acid 214 of Halo57 (SEQ ID NO:2) and thesubstituted sequence expressed and tested in a cell.

Using methods set forth in Example 1, a double mutant that included aK→R substitution and a W→F substitution made at the positionscorresponding to amino acid 200 and 214, respectively, of Halo57 (SEQ IDNO:2) was made and the substituted sequence expressed in a cell andphotocurrent measured.

Using methods set forth in Example 1, mutations were made in thesequence of the Natromonas pharaonis halorhodopsin (also referred toherein as Halo). Mutations included a single substitution K215R, asingle substitution W229F, and double substitution of [K215R+W229F]. Thesubstituted sequences were expressed in cells and the photocurrentmeasured. Photocurrent was used as a measure of the ion currents in acell when illuminated by light in voltage-clamp mode. Because a cell'shyperpolarization is induced by the light-activated ion influx,photocurrent is directly correlated to the degree of cell polarization.

Results

Results, shown in FIG. 6A, showed that the photocurrent of the Halo57mutant that included the single substitution K200R was increased ascompared to the photocurrent of the wild-type Halo57 LAIP. Thephotocurrent of the mutant that included the single substitution W214Fshowed a decrease in the photocurrent compared to the wild-type Halo57LAIP. The double substitution of K200R+W214F resulted in a synergisticeffect on the photocurrent, with the resulting photocurrentsignificantly greater than either the wild-type Halo57 LAIP or either ofthe single substitutions.

Results of the experiments, shown in FIG. 6B, showed results comparingthe effects of mutations on Halo versus Halo57. For each pair of bars,the bar on the left is Halo and the bar on the right is Halo57. Themutation number below the bars represents the mutation positionsrelative to the Halo sequence. The corresponding amino acid residues inthe Halo57 sequence (SEQ ID NO:2) are Halo57 K200R substitutioncorresponds to Halo K215R substitution and the Halo57 W214F substitutioncorresponds to Halo W229F substitution.

The results showed that inclusion of the Halo57 K200R and Halo K215Rsubstitution increased photocurrent significantly more in Halo57 than inHalo. The Halo57 W214F and Halo W229F substitution both reduced thephotocurrent compared to the Halo57 and Halo wild-type photocurrent,respectively. The Halo57 K200R+W214F and Halo K215R+W229F had verydifferent effects. In Halo57, the K200R+W214F double substitutionresulted in a synergistic effect boosting the photocurrent significantlyover the wild-type level or that of either single substitution. Incontrast, in Halo, the K215R+W229F double substitution resulted in avery low photocurrent, which was significantly lower than even the Halowild-type photocurrent level. In FIG. 6B, the first bar of each pair isHalo, and the second bar is Halo57. All of the values were normalizedrelative to the wild-type (mutant/wildtype). The experiment demonstratedthe significantly different impact of the substitutions on Halo andHalo57 photocurrents. The left-most pair of bars shows Halo (left) andHalo57 (right) wild-type normalized to themselves (i.e., 1). The secondpair of bars from left show that K215R dropped Halo photocurrents butthe corresponding substitution in Halo57 (K200R) boosted Halo57 byapproximately 70%. The third pair of bars from left show that W229F(equivalent=W214F in Halo57) lowered both Halo and Halo57, and finallythe far right pair of bars shows that the double mutation significantlylowered Halo photocurrent and substantially boosted Halo57 photocurrent.

Example 3 Methods

Trafficking of LAIP sequences was examined using various signal andexport sequences. Using methods set forth in Example 1, various signaland export sequences were included in vectors along with the LAIPsequence of Halo57 with a K200R+W214F double substitution. Included werethe following single sequences or combined sequences:

“ss”, having a nucleic acid sequence set forth herein as SEQ ID NO:15encoding the amino acid sequence set forth herein as SEQ ID NO:16.

“ER2”, having a nucleic acid sequence set forth herein as SEQ ID NO:19encoding the amino acid sequence set forth herein as SEQ ID NO:10.

“ss-prl”, having the nucleic acid sequences set forth herein as SEQ IDNO:15 and SEQ ID NO:17 encoding the amino acid sequence set forth hereinas SEQ ID NO:16 and SEQ ID NO:18.

“ss-ER2”, having the nucleic acid sequences set forth herein as SEQ IDNO:15 and SEQ ID NO:19 encoding the amino acid sequence set forth hereinas SEQ ID NO:16 and SEQ ID NO:20.

“Prl-ER2”, having the nucleic acid sequences set forth herein as SEQ IDNO:17 and SEQ ID NO:19 encoding the amino acid sequence set forth hereinas SEQ ID NO:18 and SEQ ID NO:20.

“ss-prl-ER2, including the nucleic acid sequences set forth herein asSEQ ID NO:15, SEQ ID NO:17, and SEQ ID NO:19, encoding the amino acidsequence set forth herein as SEQ ID NO:16, SEQ ID NO:18, and SEQ IDNO:20.

“Kir2.1 (KGC-ER2), having the nucleic acid sequences set forth herein asSEQ ID NO:21 and SEQ ID NO:19 encoding the amino acid sequence set forthherein as SEQ ID NO:22 and SEQ ID NO:20.

A control LAIP having no trafficking or export sequences was alsotested. The photocurrent was determined for each of the above LAIPconstructs.

Results

The results illustrated in FIG. 7 show the photocurrent effects obtainedusing signal sequences to boost membrane trafficking of light-activatedion pumps of the invention, as assessed in primary hippocampal mouseneuron culture via whole-cell patch claim. Each bar demonstrates thedifferent amounts of photocurrent generated when illuminated with 570/50nm light.

Example 4

Cell silencing was examined using LAIPs. Silencing using red light wastested in an LAIP Halo57 with a double substitution K200R+W214Fexpressed in a cell. Methods of preparation, expression and measurementwere as set forth in Example 1 with average spike rates determined. FIG.8 shows the average spike rate of a cell that includes a LAIP having thesequence of Halo57 set forth herein as SEQ ID NO:2 with a doublesubstitution K200R+W214F. The amino acid sequence of Halo57 with theK200R+W214F substitution is set forth herein as SEQ ID NO:26. FIG. 8shows results of an in vivo implementation contacting thelight-activated ion pump with a light at 655 nm. The top trace is anextracellular recording showing the cell silencing during the fiveseconds of light contact, and the bottom trace shows the averagereduction in spike frequency over a total of seven trials.

Example 5 Methods

Studies were performed to examine effects of mutagenesis of residues inHalo57 delivered using vectors that also included combinations of signaland export sequences. The efficacy of the combinations were tested usingmethods described in Example 1. The photocurrents of (1) Halo, (2)Halo57 with the ss-prl-ER2 sequence (see Example 4), (3) Halo57 havingthe double substitution K200R+W214F, and with the ss-prl-ER2 sequence;and (4) Halo57 having the double substitution K200R+W214F and with theKir2.1 sequence were determined. The sequence of ss-prl-ER2 includesnucleic acid sequences set forth herein as SEQ ID NO:15, SEQ ID NO:17,and SEQ ID NO:19, encoding the amino acid sequence set forth herein asSEQ ID NO:16, SEQ ID NO:18, and SEQ ID NO:20. The sequence of Kir2.1includes nucleic acid sequences set forth herein as SEQ ID NO:21 and SEQID NO:19 encoding the amino acid sequence set forth herein as SEQ IDNO:22 and SEQ ID NO:20.

Results

Results shown in FIG. 9 illustrate effects of mutagenesis of residues inHalo57. The results shown are from combined trafficking and mutationstudies. A combination of protein trafficking enhancements with directedmutagenesis resulted in extremely powerful red-light drivable neuralsilencers as seen for the Halo57 and Halo57 mutants.

Example 6

LAIPs of the invention were prepared and tested using methods set forthin Example 1. Various mutated sequences were prepared, expressed incells, and tested using methods described in Example 1. In particular,the A122D, K200R and W214F mutations to cruxhalorhodopsins andadditionally, the Natromonas pharaonis halorhodopsin, were found boosteffective light sensitivity and photocurrent amplitude to hyperpolarizeexcitable cells.

Numerous substitutions and combinations of substitutions were made inhaloarcula and halomicrobium, and other members of the Halobacteriaceaefamily. Single and multiple substitutions that were made to thesequences, which were then expressed and tested in cells. Substitutionsincluded: K200R, T111S, K200H, K200Q, T203S, [K200Q+W214F],[K200H+W214F], and were made in sequences including Halo57, Gene4,Gene58, Gene 55, Gene55, and Gene54, with the amino acid identificationbased on alignment of the sequences with Halo57, with amino acids thatcorresponded to the Halo57 substituted amino acids, substituted in theother, aligned sequences. The mutation position information above isgiven in reference to Halo57 sequence set forth here as SEQ ID NO:2.Each of the tested mutations was found to boost photocurrent compared tothe photocurrent of a non-substituted Halo57 LAIP.

Example 7

Genes described under (a), (b) and (c) were expressed in cells usingmethods provided below.

Genes

The Halobacterium salinarum (strain shark) gene for halorhodopsinreferred to herein as Halo57 and having the amino acid sequence setforth herein as SEQ ID NO:2 and a human codon-optimized DNA sequence setforth herein as SEQ ID NO:1;

b) The gene for Halobacterium salinarum (strain port) referred to hereinas Halo58 and having the amino acid sequence set forth herein as SEQ IDNO:4 and a mammalian codon-optimized DNA sequence set forth herein asSEQ ID NO:3; and

c) The gene for Haloarcula marismortui cruxhalorhodopsin referred toherein as Gene4 and having the amino acid sequence set forth herein asSEQ ID NO:6 and having a mammalian codon-optimized DNA sequence setforth herein as SEQ ID NO:5 are expressed in cells as follows.

Methods

(1) The opsin gene was cloned into a lentiviral or adeno-associatedvirus (AAV) packaging plasmid, or another desired expression plasmid. Insome tests GFP was cloned downstream of the preferred gene, eliminatingthe stop codon of the opsin gene, thus creating a fusion protein. Insome tests no fluorophore was included. In some tests a fusion proteinis not utilized and an IRES-GFP.

(2) The viral or expression plasmid was prepared that contained either astrong general promoter, a cell-specific promoter, or a strong generalpromoter followed by one or more logical elements (such as alox-stop-lox sequence, which would be removed by Cre recombinaseselectively expressed in cells in a transgenic animal, or in a secondvirus, thus enabling the strong general promoter to then drive thegene).

(3) When a viral plasmid was used, the viral vector was synthesizedusing the viral plasmid.

(4) If using a virus, as appropriate for gene therapy (over 600 peoplehave been treated with AAV carrying various genetic payloads to date, in48 separate clinical trials, without a single adverse event), the virusis injected using a small needle or cannula into the area of interest,thus delivering the gene encoding the opsin fusion protein into thecells of interest. If using another expression vector, the vector isdirectly electroporated or injected into the cell or organism (foracutely expressing the opsin, or making a cell line, or a transgenicmouse or other animal).

(5) Products prepared using sections 1-4 were illuminated with light.Peak illumination wavelength in some experiments was 604 nm whenincident intensity was defined in photons/second.

(6) To illuminate two different populations of cells (e.g., in a singletissue) with two different colors of light, one population is firsttargeted with a haloarcula such as the Halobacterium salinarum (strainshark)/Halobacterium halobium (strain shark) gene for halorhodopsin, andthe other population is targeted with a blue-shifted opsin such as Mac(3), using two different viruses (e.g., with different coat proteins orpromoters) or two different plasmids (e.g., with two differentpromoters). Then, after the molecule expresses, the tissue isilluminated with 450±25 nm, 475±25 nm, or 500±25 nm light topreferentially hyperpolarizing the Mac-expressing cells, and the tissueis illuminated with 660±25 nm light to preferentially hyperpolarize theHalobacterium salinarum (strain shark)/Halobacterium halobium (strainshark) halorhodopsin-expressing cells. The above wavelengths illustratetypical modes of operation, but are not meant to constrain the protocolsthat can be used. Either narrower or broader wavelengths, ordifferently-centered illumination spectra, can be used. For prostheticuses, the devices used to deliver light may be implanted. For drugscreening, a xenon lamp or LED can be used to deliver the light.

DISCUSSION

According to principles of this invention, the performance of the abovesaid example compositions of matter may be altered by site-directedmutagenesis, such as the A137D single mutation, the K215R singlemutation, and the W229F single mutation to Halo, and the A122D singlemutation to the haloarcula class, the K200R single mutation to thehaloarcula class, and the K200R+W214F double mutation to the haloarculaclass (see FIG. 3). Also, according to principles of this invention, theperformance of the above may be altered by appending N-terminal andC-terminal peptide sequences to affect cellular trafficking, such as theN-terminal prolactin endoplasmic sorting sequence (denoted as ‘PRL’)(amino acid sequence: MDSKGSSQKGSRLLLLLVVSNLLLCQVVS (SEQ ID NO:18); DNAsequence:gacagcaaaggttcgtcgcagaaagggtcccgcctgctcctgctgctggtggtgtcaaatctactcttgtgccagggtgtggtctccacccccgtc; (SEQ ID NO:17), or the MHC class I antigen signal sequence(denoted as “ss”) (amino acid sequence: MVPCTLLLLLAAALAPTQTRA (SEQ IDNO:16); DNA sequence:gtcccgtgcacgctgctcctgctgttggcagccgccctggctccgactcagacgcgggcc (SEQ IDNO:15), or the C terminal Kir2.1 signal sequence (denoted as “ER2”)(amino acid sequence: FCYENEV (SEQ ID NO:20); DNA sequence:ttctgctacgagaatgaagtg (SEQ ID NO:19)), or the C terminal Kir2.1 signalsequence (denoted as “KGC”) (amino acid sequence: KSRITSEGEYIPLDQIDINV(SEQ ID NO:22); DNA sequence:aaatccagaattacttctgaaggggagtatatccctctggatcaaatagacatcaatgtt (SEQ IDNO:21)), or combinations thereof, as exemplified by the ss-Prl-Arch(i.e. ss::prl::Arch fusion) molecule (Genbank accession # GU045597), orss-Prl-Arch-GFP (Genbank accession # GU045599).

In some studies, this invention uses light-activated chloride pumps tohyperpolarize neurons. Haloarcula-derived cruxhalorhodopsins arered-light-drivable, which allows hyperpolarization of cells with a colorof light heretofore not predominately used in biotechnology forhyperpolarization of cells. By using the Halobacterium salinarum (strainshark)/Halobacterium halobium (strain shark) gene for halorhodopsin inconjunction with presently existing tools such as Mac, hyperpolarizationof two different populations of cells in the same tissue or in the sameculture dish becomes possible with substantially less cross-excitationinterference seen with less red light driven opsins such as thepresently existing Halo. This simultaneous, two-color inactivation, isparticularly promising for complex tissues such as the brain.Multi-color perturbation is not limited to only two colors, nor must theperturbation be of the same physiological function.

According to principles of this invention, the performance of the abovesaid molecules or classes of molecules are tuned for optimal use,particularly in context of their use in conjunction with other moleculesor optical apparatus. For example, in order to achieve optimal contrastfor multiple-color silencing, one may either improve or decrease theperformance of one molecule with respect to one another, by theappendage of trafficking enhancing sequences or creation of geneticvariants by site-directed mutagenesis, directed evolution, geneshuffling, or altering codon usage.

In some studies, the haloarcula genus is used. These have beenidentified as particularly efficacious light-activated chloride pumpsbecause they express particularly well in mammalian membranes andperform robustly under mammalian physiological conditions.

Example 8

Aspects of the invention include compositions of matter that have beenreduced to practice, as described below:

Plasmids encoding for the above genes, have been prepared and used todeliver genes into cells, where the genes have been expressed. As anexemplary vector, lentiviruses carrying payloads encoding for the abovegenes have been prepared and used to deliver genes into cells resultingin expression of the LAIP in the cells. In addition, adeno-associatedviruses carrying payloads encoding for the above genes have beenprepared and used to deliver genes into cells, resulting in theexpression of the LAIP in the cells. Cells have been prepared thatexpress the LAIP genes set forth in Example 7 and those of FIG. 3.Animals have been prepared that include cells that express the LAIPgenes set forth in Example 7, and those of FIG. 3.

It is to be understood that the methods, compositions, and apparatuswhich have been described above are merely illustrative applications ofthe principles of the invention. Numerous modifications may be made bythose skilled in the art without departing from the scope of theinvention.

Although the invention has been described in detail for the purpose ofillustration, it is understood that such detail is solely for thatpurpose and variations can be made by those skilled in the art withoutdeparting from the spirit and scope of the invention which is defined bythe following claims.

The contents of all literature references, patents, and published patentapplications cited throughout this application are incorporated hereinby reference in their entirety.

We claim:
 1. A vertebrate cell comprising a light-activated ion pumppolypeptide that when expressed in a mammalian excitable cell andcontacted with a red light silences the excitable cell, wherein thepolypeptide sequence of the light-activated ion pump comprises an aminoacid sequence set forth herein as SEQ ID NO:2, with one, two, or moreamino acid sequence modifications, and wherein the polypeptide has atleast 70% amino acid identity to the sequence set forth as SEQ ID NO:2.2. The vertebrate cell of claim 1, wherein the polypeptide sequencecomprises one or more of: a) a K→R, K→H, or K→Q substitution at an aminoacid residue corresponding to amino acid 200 of the amino acid sequenceof Halo57 (SEQ ID NO:2); b) a T→S substitution at an amino acid residuecorresponding to amino acid 111 of the amino acid sequence of Halo57(SEQ ID NO:2); c) a T→S substitution at an amino acid residuecorresponding to amino acid 203 of the amino acid sequence of Halo57(SEQ ID NO:2); or d) a K→Q+W→F double substitution at the amino acidresidues corresponding to amino acid 200 and 214, respectively, of theamino acid sequence of Halo57 (SEQ ID NO:2).
 3. The vertebrate cell ofclaim 1, wherein the vertebrate cell is an excitable cell.
 4. Thevertebrate cell of claim 1, wherein the vertebrate cell is a mammaliancell.
 5. The vertebrate cell of claim 1, wherein the vertebrate cell isin vitro, ex vivo, or in vivo.
 6. The vertebrate cell of claim 1,further comprising one, two, three, four, or more additionallight-activated ion pumps, wherein at least one, two, three, four, ormore of the additional light-activated ion pumps is activated by contactwith light having a non-red light wavelength.
 7. A method of identifyingan effect of a candidate compound on a vertebrate cell, the methodcomprising, a) contacting a vertebrate test cell comprising alight-activated ion pump polypeptide that when expressed in a mammalianexcitable cell and contacted with a red light silences the excitablecell, wherein the polypeptide sequence of the light-activated ion pumpcomprises an amino acid sequence set forth herein as SEQ ID NO:2, withone, two, or more amino acid sequence modifications, and wherein thepolypeptide has at least 70% amino acid identity to the sequence setforth as SEQ ID NO:2, with a light under conditions suitable to activatethe ion pump and hyperpolarize the test cell; b) contacting thevertebrate test cell with a candidate compound; and c) identifying thepresence or absence of a change in the hyperpolarization or in ahyperpolarization-mediated cell characteristic in the vertebrate testcell contacted with the light and the candidate compound compared to thehyperpolarization or the hyperpolarization-mediated cell characteristic,respectively, in a control vertebrate cell contacted with the light andnot contacted with the candidate compound; wherein a change in thehyperpolarization or the hyperpolarization-mediated cell characteristicin the vertebrate test cell compared to the vertebrate control cellidentifies an effect of the candidate compound on the vertebrate testcell.
 8. The method of claim 7, wherein the effect of the candidatecompound is an effect on the hyperpolarization of the vertebrate testcell.
 9. The method of claim 7, wherein the effect of the candidatecompound is an effect on a hyperpolarization-mediated cellcharacteristic in the vertebrate test cell.
 10. The method of claim 7,wherein the hyperpolarization mediated-cell characteristic is ahyperpolarization-activated conductance.
 11. The method of claim 10,wherein the hyperpolarization-activated conductance is the result of aT-type calcium channel activity, a BK channel activity, or an I_hcurrent.
 12. The method of claim 7, wherein the hyperpolarizationmediated-cell characteristic is cell silencing.
 13. The method of claim7, wherein the modified polypeptide sequence comprises one or more of:a) a K→R, K→H, or K→Q substitution at an amino acid residuecorresponding to amino acid 200 of the amino acid sequence of Halo57(SEQ ID NO:2); b) a T→S substitution at an amino acid residuecorresponding to amino acid 111 of the amino acid sequence of Halo57(SEQ ID NO:2); c) a T→S substitution at an amino acid residuecorresponding to amino acid 203 of the amino acid sequence of Halo57(SEQ ID NO:2); or d) a K→Q+W→F double substitution at the amino acidresidues corresponding to amino acid 200 and 214, respectively, of theamino acid sequence of Halo57 (SEQ ID NO:2).
 14. The method of claim 7,wherein the modified polypeptide sequence comprises a K→R modificationand a W→F modification at the amino acid residues corresponding to aminoacid 200 and 214, respectively, of the amino acid sequence of Halo57(SEQ ID NO:2).
 15. The method of claim 7, wherein the modifiedpolypeptide sequence is the sequence set forth as SEQ ID NO:26.
 16. Themethod of claim 7, wherein the vertebrate test cell is a nervous systemcell, a neuron, a cardiac cell, a circulatory system cell, a visualsystem cell, an auditory system cell, a hemoglobin-rich cell, or amuscle cell.
 17. The method of claim 7, wherein the vertebrate test cellis an excitable cell, and optionally is a mammalian cell.
 18. The methodof claim 7, wherein the vertebrate test cell is a mammalian cell. 19.The method of claim 7, wherein the vertebrate test cell furthercomprises one, two, three, or more additional light-activated ion pumps,wherein at least one, two, three, four, or more of the additionallight-activated ion pumps is activated by contact with light having anon-red light wavelength.
 20. An isolated nucleic acid sequence thatencodes a light-activated ion pump polypeptide that when expressed in amammalian excitable cell and contacted with a red light silences theexcitable cell, wherein the polypeptide sequence of the light-activatedion pump comprises an amino acid sequence set forth herein as SEQ IDNO:2, with one, two, or more amino acid sequence modifications, andwherein the polypeptide has at least 70% amino acid identity to thesequence set forth as SEQ ID NO:2.